Downy mildew disease, caused by the obligate pathogen Peronospora effusa, is the most economically important disease of spinach. Fourteen of the 17 named races and 11 novel strains of P. effusa have been identified in the past 30 years. However, the mechanism of the rapid evolution of virulence remains unknown. A total of 69 downy mildew isolates (an isolate is defined as a collection of nearby symptomatic leaves within a given field) were collected from 14 states from 2010 to 2018. The genotypes of between 1 and 39 individual lesions per isolate were examined using a targeted sequencing technique to sequence 33 SNP loci. Genetic diversity was evaluated between individual lesions within a given isolate, between fields, host cultivars, geographic locations, and years. A total of 387 multilocus SNP genotypes (MLG) were identified among 731 sequence samples examined. Among the 387 genotypes, 315 (81%) were unique. The most common genotype (MLG374) was identified in 110 sequence samples from 16 isolates collected from CA and AZ in 2016. The variation for a given isolate ranged from no variation (a single genotype among lesions from that isolate) to many genotypes (highest 37) from lesions of that isolate. An index of association analysis indicated evidence for both asexual (clonal) and sexual reproduction. Four subpopulations were found by discriminate analysis of principal components. Host cultivar, origin and time of collection had effects on population differentiation, and genotypes specific to certain location or time period were identified.