Livestock production is a key element for poverty alleviation, food security, and economic growth in Rwanda. In 2017, the national average milk production per cow was about 2.5 L per day; in 2020-2021, it is projected to increase to 3.5 L per day if improvement interventions including those designed to reduce the burden of brucellosis in cattle are implemented. The objective of the study reported here was to estimate the seroprevalence of and identify risk factors associated with dairy farms and cattle classified as seropositive to Brucella spp. in three different agroecological zones in Rwanda. Most study farms (40/85 or 47%) had one head of cattle only. Using the Rose Bengal test, the seroprevalence of brucellosis was 28/85 or 33% (95% CI = 24%, 43%) at the farm level and 63/465 or 14% (95% CI = 11%, 17%) at the animal level. Using logistic regression, at the farm level, the presence of seropositive cattle was associated with herd size (2-45 cattle, odds ratio = 21.2; 95% CI = 2.4, 184.5) (46-220 cattle, OR = 288.5; 95% CI = 24.3, 3,423.1) compared to farms with one animal, after controlling for main breed (local breeds, crossbreeds) on the farm. In addition, the odds of testing seropositive were 10.7 (95% CI = 2.3, 49.1) and 149.5 (95% CI = 19.3, 1,158.7) times higher in farms in Nyabihu district and Nyagatare district, respectively, than in farms in Muhanga district, after controlling for main breed on the farm. The odds of seropositivity to Brucella spp. were 2.8 times higher in farms with mostly local breeds, than in those with mostly crossbreeds; but the association was confounded by herd size and geographic location. At the animal level, the odds of seropositivity to Brucella spp. were 2.6 times higher in adult cattle than in young cattle (95% CI = 1.1, 6.3). Finally, we observed a high frequency of adult cattle (86%) and a high seroprevalence of brucellosis in adult cattle (25%) in Nyagatare; an indication that, in the absence of culling and other control measures, Brucella spp. infection pressure can be relatively constant and a steady source of disease transmission in pastoral systems in that district.