We developed PCR-based assays to distinguish a human pathogenic strain of Anaplasma phagocytophilum, Ap-ha, from Ap-variant 1, a strain not associated with human infection. The assays were validated on A. phagocytophilum-infected black-legged ticks (Ixodes scapularis) collected in Canada. The relative prevalence of these 2 strains in I. scapularis ticks differed among geographic regions.