Onion is one of the most important economic and dietary crops belonging to Liliaceae that widely cultivated in all over the world. Among the important diseases of onion، bacterial diseases such as Pectobactrium soft rot ( Pectobacterium sp.)، Dickeya soft rot ( Dickeya chrysanthemi ) and slippery skin ( Burkholderia gladioli ) occur frequently worldwide. In recent years، onion soft rot disease caused great damages in onion producing area of Isfahan provinces. Based on main symptoms of the diseases including yellowing and wilting of aerial parts and soft rot of onion bulbs in farms and post-harvest stages، it was inferred that likely bacteria are the causal agents of this important disease. In order to identify and characterize bacterial causal agents of the disease، sampling was done from onion farms with soft rot symptom in Fereydan، Flavarjan and Ghahderijan. By culturing in general and specific media، 57 bacteral isolates were obtained from the collected samples. Fingerprinting of the isolates by rep-PCR method using BOX1AR primer generated four fragments in size of 200 to 1000 bp with different band patterns. Based on BOX- banding patterns، the isolates were differentiated into two groups I and II. According to morphological، pathological، and biochemical characteristic and also pectolytic activity، the isolates of group I placed in two subgroup Ia and Ib. The isolates of subgroup Ia، including 37 of 57 isolates، because of positive reaction of phosphatase، indole and acid production from lactose، mannitol and negative reaction for reducing substances from sucrose، utilization of trehalose، ?- methyl D-glucoside، dulcitol and sorbitol، characterized as D. chrysanthemi ( Dch ). Other 11 isolates belonging to Ib group that in terms of some biochemical characteristic such as acid production from lactose and lack of production phosphatase، indole and reducing substances from sucrose identified as P. carotovorum su. carotovorum ( Pcc ). In order to ascertain species identification، all isolates were used as template in PCR reactions. Using species specific primer pairs Y1/Y2 and ExpccF/ExpccR amplified a 434 bp and 550 bp fragments in the isolates identified as Pcc (Ib). The results of sequencing of the latest fragment indicated 96% identity of isolates subgroup Ib with sequences of GenBank isolates. The isolates introduced as D. chrysanthemi (Ia) amplified 420 and 1263 bp fragments by using primer pairs ADE1/ADE2 and pelZ-1-F/pelZ-1-R. Nucleotide sequences and alignment of amplicon 420bp with available sequences in GenBank، determined 97% sequences similarity of subgroup Ia representetives with Dch isolates in GenBank. Using primer pairs G1/L1 in PCR reaction، ITS region of isolates amplified and based on banding pattern، they are placed in different two groups. Bacterial isolates of group II belonging to post-harvest bulb onion samples produced yellow-brown، wrinkled and serrated margins colonies in NA medium. The results of pathogenicity and biochemical tests of group II isolates revealed that these isolates belong to Burkholderia gladioli . Also، these isolates amplified a 468 bp fragment in PCR reaction using primer pairs CMG-16-1/CG-16-2 containing nucleotide sequences similar to B. gladioli . According to the results of this study، it was determined that P. carotovorum su . carotovorum and D. chrysanthemi are causal agents of onion soft rot in fields of Isfahan provinces and B. gladioli is involved in onion bulb rot in post-harvest stages.