Serologic tests currently available for brucellosis diagnosis detect antibodies to Brucella but do not distinguish between species of Brucella. Although Brucella suis is known to circulate within various feral swine (Sus scrofa) populations, our objective was to determine the primary species of Brucella circulating in feral swine populations in areas of the US with high brucellosis prevalence. We cultured lymph nodes from 183 feral swine. We identified 22 isolates from 21 animals, and all isolates were genotyped as B. suis. Most isolates were B. suis biovar 1, with the exception of two genetically distinct isolates from one feral swine in Hawaii, which were identified as B. suis biovar 3. Serum from each feral swine was also tested by the fluorescence polarization assay when possible, but only 52% (95% CL = 29.8–74.3) of culture-positive animals were antibody positive. Our results indicate that brucellosis infections in feral swine within the US are typically caused by B. suis. However, improved serologic tests are needed to more accurately determine exposure to Brucella spp. and to monitor disease trends in feral swine populations.