Brucellosis is a neglected zoonotic disease affecting the livestock sector in low‐income countries. Cameroon, a lower‐middle‐income country in sub‐Saharan Africa, has reported the prevalence of brucellosis in regions where livestock rearing is the principal economic activity. However, the presence of the disease has not been reported in southern regions receiving cattle for consumption by their population. In addition, there is no report on the Brucella species circulating in Cameroon. This study aimed to determine the prevalence of brucellosis in cattle slaughtered in the Buea and Douala slaughterhouses and identify the Brucella species circulating among these animals. A total of 576 cattle serum samples were collected from the Buea and Douala slaughterhouses and analysed by ELISA. Following the ELISA assay, all samples were subjected to polymerase chain reaction (PCR) analysis. The bcsp31 gene primers were used for the genus‐specific PCR. All bcsp31‐positive samples were subjected to species‐specific PCR. Primers targeting the IS711 gene sequence were used to identify the abortus‐melitensis‐ovis‐suis species. The prevalence of brucellosis in both locations was 3.1% and 5.4% using the ELISA and PCR assays, respectively. Out of the 18 ELISA‐positive samples, 5 (27%) were positive with PCR, while 26 (4.7%) of the ELISA‐negative samples were positive with PCR. The Brucella species in circulation were identified using the Sanger sequencing technique. The sequences were 99.3% to 100% identical to the B. abortus strain BJ1‐23 and the B. abortus strain BJ1‐1 of the 31 kDa antigen (bcsp31) gene from India. This is the first report on the genotypic characterisation of Brucella species in Cameroon and confirms brucellosis in cattle at the Buea and Douala slaughterhouses.