Genetic diversity and biological variation were compared for California isolates of Cucumber mosaic virus (CMV). These fell into five pathotypes based on their reactions on three cucurbits including a susceptible squash, a melon with conventional resistance and a commercial CMV-resistant transgenic squash. Thirty-three isolates infected and caused symptoms on CMV-resistant transgenic squash. Forty-two isolates infected the CMV-resistant melon, but only 25 isolates infected both. Single-strand conformation polymorphism (SSCP) analysis was used to differentiate 81 California isolates into 14 groups, and the coat protein (CP) genes of 27 isolates with distinct and indistinguishable SSCP patterns were sequenced. Fourteen isolates corresponding to the different SSCP patterns were also used for phylogenetic analysis. Seventy-nine isolates belonged to CMV subgroup IA, but two belonged to CMV subgroup IB. This is the first report of subgroup IB isolates in the Americas. All CMV isolates had a nucleotide identity greater than or equal to 93.24 %. There was no correlation between CP gene variation and geographical origin, collection year, original host plant, or between the degree of CP amino acid sequence identity and the capacity to overcome transgenic and/or conventional resistance. SSCP and sequence analyses were used to compare 33 CMV isolates on CMV-resistant transgenic squash and susceptible pumpkin plants. One isolate showed sequence differences between these two hosts, but this was not due to recombination or selection pressure of transgenic resistance. CMV isolates capable of infecting cucurbits with conventional and transgenic CMV resistance were present in California, even before CMV transgenic material was available.