The present study was conducted to evaluate nitrogen use and translocation efficiency, photoassimilates distribution and allelopathic functions of ancient wheats of different ploidy levels in response to nitrogen in the field, pot and laboratory experiments. A two-year field experiment (3-replicate factorial randomized complete block design) was conducted on five hulled tetraploid emmer wheat genotypes and one improved free-threshing durum along with a bread wheat. Moreover, a pot experiment (3-replicate factorial completely random design) was carried out on 12 genotypes encompassing wheats from all ploidy levels (i.e. di, tetra, and hexaploid) and the ancient hulled vs the improved durum and bread wheats during the 2016–2017 and 2017–2018 growing seasons. Nitrogen (N) fertilizer was applied to the examined wheats at the wide-ranging 0-75 (field experiment) and 0-120 (pot experiment) kg ha -1 N rates to achieve various N supplies/deficiencies.Interacting effect of wheat genotype × N and was statistically significant for all measured traits. Leaf chlorophyll and carotenoids concentrations of all wheat types were increased in response to N supply, but the extent of increases was smaller for the ancient emmer and spelt wheat types. Yield components (i.e. spikes/plant, seeds/spike, 1000-seeds weight), grain yield, DM, and harvest index of the standard durum and bread wheats tended to increase with increase in the N supply, but those of the ancient wheats were increased when exposed to the moderate N and decreased when grown in the presence of the high N supply. Photoassimilates translocation of the ancient emmer wheats was different from that of the standard durum and bread wheats in terms of modifications in response to N supply, resulting in smaller amounts of photoassimilates partitioned to the grains and, hence, lower harvest indices of the ancient wheats, in general, and in the presence of the high N supply, in particular. Though, the ancient wheats, in general, and emmer wheats, in particular, tended to indicate greater or comparable N uptake and utilization in comparison to improved durum and bread wheats, when exposed to N deprivation. In the laboratory experiment, wheat genotypes, which were treated with nitrogen fertilizer in the field experiment, were harvested at pollination stage (BBCH SCALE 65). The plant samples were dried in the shade and transferred to the laboratory for bioassay. The plant samples were milled and four concentrations of aqueous extract (2.5, 5, 7.5 and 10%) were prepared. Laboratory experiment (3-replicate factorial completely random design) consisted of two allelopathic experiments. In the first allelopathic experiment, the effect of nitrogen on the allelopathic capability of seven wheat genotypes on germination and seedling growth of radish ( Raphanus sativus L.) as a test plant was investigated. In the second allelopathic experiment, the effect of nitrogen on allelopathic properties of four wheat genotypes, selected from the first allelopathic experiment, on germination, seedling growth and photosynthetic pigments of wild barley ( Hordeum vulgare . spontaneum L.) was investigated. In both experiments biological screening of wheat genotypes demonstrated that all wheat genotypes suppressed the seedling growth of Raphanus sativus L. and Hordeum vulgare . spontaneum L. Modern hexaploid wheat genotype along with ancient tetraploid emmer wheat genotypes in all nitrogen fertilizer treatments possessed a high allelopathic capability. Allelopathic capability of the ancient emmer wheats was increased when exposed to the low and medium N supplies. However, the allelopathic capability of the standard durum and bread wheats was increased with increases in the N supply. High performance liquid chromatography diode array detection analysis of aqueous extractrevealed that these genotypes differed considerably in their ability to synthesis syringic acid. The total amount of polyphenolics and flavonoids compounds in shoot aqueous extract showed a significant positive correlation to the R. sativus and H. spontaneum growth suppression. These results suggest that the number of polyphenolics in general and syringic acid in particular might have contributed to the interfering ability of ancient tetraploids and modern hexaploid wheat genotypes. At the same time, results showed that ancient tetraploid emmer wheats may offer promising values for their unique allelochemicals content, denoting their application in sustainable crop production systems. Key words Ancient wheat, Dry matter translocation, N utilization; Allelopathy, Polyphenolics