Brucellosis is a key zoonosis of major public health, animal welfare, and economic significance, and is endemic in livestock in Uganda. A cross-sectional epidemiological study was carried out to estimate the sero-prevalence of brucellosis and identify factors associated with sero-positivity in cattle in urban and peri-urban Gulu and Soroti towns of Northern and Eastern Uganda, respectively. A total of 1007 sera and data on biologically plausible risk factors from 166 herds and their spatial locations, were collected from cattle reared in urban and peri-urban Gulu and Soroti towns of Uganda. The sera were analyzed using indirect ELISA and sero-positive reactors confirmed by competitive ELISA. Multivariable models were used to investigate for risk factors. The overall animal-level and herd level sero-prevalence was 7.5% (76/1007, 95% Confidence Interval (CI): 6.15 – 9.4%) and 27.1% (45/166, 95% CI: 20.9% - 34.3%), respectively. Herd-level sero- prevalence was significantly (p<0.001) higher in Soroti than Gulu. In Gulu town, sero- positivity increased with an increase in herd size (p=0.03) and age (p=0.002), and was higher in cattle brought in from western Uganda (p<0.0001). In Soroti town, introduction of new cattle into a herd was significantly (p=0.027) associated with herd sero-positivity. There was a geographically differential risk (clustering) of Brucella sero- positivity in herds in Soroti, while sero-positivity was homogeneously distributed in Gulu. The data highlights brucellosis occurrence and major risk factors for its transmission in cattle in urban and peri-urban areas.