In recent years, onion sour and watery skin disease has been emerged frequently in field grown onions and their stored bulbs. Yellowing and colapsing of leaves of the infected plants, softening of the plant neck, water soaking, souring and discoloration of the skins to gray are the main symptoms of the disease in onion fields and stored onion bulbs. In order to identify the causal agent of the disease, sampling was done from onion fields of Zeyar, Falavarjan, Ghahdrijan, Dameneh, Daran, Faridan and vegetable grocery stores of Khomeini-Shahr and Isfahan. By culturing the samples on nutrient media, 120 bacterial isolates were obtained including 74 from field and 46 from stored bulbs. pathogencity of the isolates were proved invitro and invivo . On the basis of rep-PCR results, using BOX and ERIC1R/ ERIC2Fprimers, all the isolates were divided into four separate groups named I(45 isolates), II (20 isolates), III (9 isolates) and IV(46 isolates). The morphological, physiological and biochemical test results were not the authentic ways for the precise identification of the species or genus of the isolates grouped as I, II and III.The members of the groups did not even produce any DNA fragment in PCR reaction using specific primer pairs of known bacteria, causing onion sour skin diseases including Burkholderia cepacia, B. gladioli, Pantoea ananatis, Pseudomonas viridiflava . Therefore, a 1480 bp fragment of the 16S rDNA region of the representative isolates from each group was amplified by using prokaryoticuniversal primers fD1/rD1. The amplicons of the isolates were sequenced and subjected to comparative sequence analysis with available sequences in Gene Bank (BLAST). Based on the results, the sequenced 16S rDNA region of the group I with large, mucuoid, irregular and translucent with central whitish dot colonies exhibited 99 to 100% similarity with known isolates of Gluconobacter frateurii . Group II isolates with pale yellow, round and flat colonies showed 99% sequence identity to the 16S rDNA gene sequence of Kosaconia cowanii and group III isolates with large, white, raised and round colonies, were highly similar (99%) to those of Entrobacter cloacae . All 46 isolates of group IV obtained from post-harvested onion bulbs were similar but not completely identical in colony morphology to isolates group III. In PCR reaction using specific primer pair for P. ananatis , Pan ITS/EC5 successfully primed the synthesis ofan expected 398 bp fragment, although the isolates were not similar in morphological characteristics to P. ananatis .The sequenced fragment showed 99% and 100% homology to the recognized Klebsiella oxycota isolates. To verify the diagnosis, the 1480 bp fragment (fD1/ rD1) of group IV isolates were sequenced and their 99% similarity to K.oxycota was confirmed. The results demonstrated that the bacteria G. frateuri , K.cowanii and E. cloacae are the causal agents of onion sour and watery skin disease in field grown onion plants in Isfahan, while K.oxycota is involved in the same disease in stored onion bulbs. All bacteria identified in this research are known as opportunistic human pathogens which can spred through sewage system into onion fields. Key words onion sour and watery skin disease, rep-PCR, Gluconobacter frateurii , Klebsiella oxycota , Kosaconia cowanii , Entrobacter cloacae