Forty‐five Iranian isolates of Xanthomonas campestris obtained from wheat, barley and grasses were compared with reference strains using polyacrylamide gel electrophoresis (PAGE) of the whole‐cell and membrane proteins. The PAGE profiles of the whole‐cell and membrane proteins of the Iranian isolates obtained from barley, with the exception of IBLS 11 and IBLS 12, were identical and clearly distinguishable from those of the other isolates. The barley group isolates, which were pathogenic only to barley, were similar to UPB 458 (NCPPB 2389), the reference strain of pathovar hordei. The isolates obtained from wheat and grasses, as well as IBLS 11 and IBLS 12, which can infect wheat, barley and some wild grasses, had similar banding patterns: only IBLS 40 isolated from Hordeum sp. displayed the same profile as the barley group. Reference strains UPB 443 (NCPPB 2821) and UPB 513, which correspond to pathovars undulosa and translucens , respectively, were related to the wheat group. IBLS45, isolated from Bromus sp., had a banding pattern that differed from those observed for strains of the barley and wheat groups. The results suggest that this method can be useful for discriminating different pathovars of X. campestris attacking cereals and grasses, and sodium dodecyl sulphate (SDS)‐PAGE of membrane proteins was not more sensitive than SDS‐PAGE of total proteins for differentiating the isolates.