Resistance to the thiocarbamates has been selected in early watergrass populations within the rice-growing region of California. To elucidate the processes contributing to the spread of resistance among rice fields, we characterized the genetic diversity and differentiation of thiobencarb-resistant (R) and thiobencarb-susceptible (S) populations across the Central Valley using microsatellite markers. A total of 406 individuals from 22 populations were genotyped using seven nuclear microsatellite primer pairs. Three analytical approaches (unshared allele, Shannon–Weaver, and allelic-phenotype statistics) were used to assess genetic diversity and differentiation in the allohexaploid species. Low levels of genetic variation were detected within populations, consistent with other highly selfing species, with S populations tending to be more diverse than R populations. FST values indicated that populations were genetically differentiated and that genetic differentiation was greater among S populations than R populations. Principal coordinate analysis generated two orthogonal axes that explained 88% of the genetic variance among early watergrass populations and differentiated populations by geographical region, which was associated with resistance phenotype. A Mantel test revealed that genetic distances between R populations were positively correlated with the geographical distances separating populations. Taken together, our results suggest that both short- and long-distance seed dispersal, and multiple local and independent evolutionary events, are involved in the spread of thiobencarb-resistant early watergrass across rice fields in the Sacramento Valley. In contrast, resistance was not detected in early watergrass populations in the San Joaquin Valley.