A cross-sectional study was conducted to determine individual cow seroprevalence of Babesia bovis in adult lactating dairy cattle of Puerto Rico (PR), to assess the associations of farm management factors on herd seroprevalence, and to document the species of ticks infesting cattle within these farms. Antibody activity against B. bovis was determined using an indirect fluorescent antibody test (IFAT). Serum samples were obtained from 2,414 adult lactating dairy cattle from 76 randomly selected commercial dairy farms. Herd seroprevalence ranged from 0 to 51% with an overall individual cow seroprevalence for B. bovis of 26%. Ticks were collected from animals on 7 (9%) of the 76 participating commercial dairy farms. All collected ticks (n = 87) were Rhipicephalus (Boophilus) microplus. Factors associated with high herd seropositivity were dairy farms with calf but not heifer raising facilities (OR = 16, 95% CI = 3.0-86), having more than 4 neighbors with cattle (OR = 17, 95% CI = 1.6-178), same producer owning more than one farm (OR = 7.2, 95% CI = 1.6-32), and use of government services to apply amitraz on cattle (OR = 5.5, 95% CI = 1.5-20).