Brucellosis has been documented in domestic water buffalo (Bubalus bubalis) but published literature is limited despite the importance of this species in tropical agricultural systems. The objective of this study was to compare the virulence of Brucella abortus isolates recovered from cattle and water buffalo. Nineteen strains of B. abortus from cattle and domestic water buffalo in Trinidad were intraperitoneally inoculated into BALB/c mice. Spleens were cultured for B. abortus and histopathological severity scores were calculated based on lymphoid depletion, lymphoid necrosis, splenitis, and macrophage accumulation. A general linear model approach was used to estimate the effect of isolate source (cattle versus water buffalo) on virulence. Isolates of water buffalo origin were significantly less virulent in the mouse model based on recovered B. abortus from splenic tissues, spleen/weight ratio, and lymphoid necrosis but not overall histopathological severity scores. Further investigation of isolates recovered from water buffalo might provide the key to the development of procedures for brucellosis control in tropical environments.