Technical Abstract: Pepino mosaic virus (PepMV) is a member of the genus Potexvirus within the family Flexiviridae. Pepino mosaic is an emerging disease in greenhouse tomato in Europe, North America and South America. Previous research in several laboratories suggests that PepMV consists of numerous sequence variants. However, the genetic composition of PepMV variants in North America has not been studied. In this report, the genetic diversity and population structure of PepMV isolates collected from several major greenhouse tomato facilities in the United States and Canada were analyzed using two pairs of primers targeting two genomic regions encoding the RNA-dependant, RNA polymerase domain of replicase, and the triple gene blocks 2 and 3 regions. In total, 84 cDNA clones derived from 31 isolates were sequenced and analyzed. All four major groups of PepMV variants were found to exist in North American greenhouse tomatoes (namely EU, US1, US2 and Ch2). Interestingly, of PepMV isolates collected from major greenhouse tomato production facilities in the U.S. and Canada, the EU (European) strain was the most dominant. In fact, PepMV isolates from Canada were homogeneous and comprised a single variant, the EU strain. In contrast, the viral population in the U.S. was much more complex, with several distinct viral variants (US1, US2, and Ch2) being identified. In PepMV viral populations recovered from mixed-infection in Arizona and Colorado, the EU strain was the most prevalent variant. A Colorado sample (CO1) obtained in 2001 possessed only the EU variant. However, in 2006, two additional genotypes (US1 and US2) were detected in Colorado. PepMV samples from Arizona were comprised of a complex of EU, US1 and US2 variants. Although all seven Texas samples acquired in 2006 were homologous for the EU strain, subsequent resampling in 2007 resulted in the detection of the Ch2 variant. The complexity of population genetics of PepMV in the U.S. poses additional challenges to the greenhouse tomato industries in virus detection and disease management.