Bovine tuberculosis (TB) is an important neglected zoonosis in Cameroon, where many communities depend on their livestock for livelihood and the incidence of human TB and TB-HIV/AIDS co-infection are high and increasing annually. The aim of this study was to estimate the prevalence of bovine TB in cattle in the highlands of Cameroon. The magnitude and trend of detecting TB lesions in slaughtered cattle (1994 to 2010) and tuberculin skin tests (TST) in 2853 cattle (84 herds) of 39 livestock rearing communities were analysed. Of 129 165 slaughtered cattle inspected, 599 (0.46%; 95% CI: 0.43%-0.50%) showed suspected TB lesions among a total of 983 (0.76%; 95% CI: 0.71%-0.81%) identified pathologies. The monthly TB detection rates ranged from 0.30% (95% CI: 0.20%-0.40%) to 0.81% (95% CI: 0.64%-0.98%) and annual rates from 0.04% (95% CI: 0%-0.11%) to 1.46% (95% CI: 1.22%-1.69%). The rates were not affected (P < 0.05) by season and fluctuating peaks were also recorded. The comparative TST revealed that bovine TB was widely distributed in live cattle (4.67%; 95% CI: 3.89%-5.44%) and was higher (χ 2 = 17.50, P ≤ 0.001) in the Western highlands than Adamawa plateaux.Comparative TST bovine TB reactors were higher (P < 0.05) in cattle managed in semi-intensive and beef production systems compared to the others. Animals in small herds showed higher (χ 2 = 4.283, P = 0.038) rates than those in large herds. Bovine TB prevalence in exotic/upgraded cattle was comparable to that of the Red Bororo zebu but higher than the rates in Guadali (χ 2 = 4.971, P = 0.026) and White Fulani (χ 2 = 5.6, P = 0.018) zebus.Among the indigenous zebus, the rate was higher in Red Bororo than the Guadali (χ 2 = 6.244, P = 0.012) and White Fulani (χ 2 = 6.568, P = 0.010). Sex did not influence (χ 2 = 0.410, P = 0.522) bovine TB prevalence in this study but diagnosis of the disease was higher (χ 2 = 5.787; P = 0.016) among adult/older cattle than in younger animals. Further analysis of the TST responses revealed that atypical mycobacterial infections was widespread and 6.83% of tested animals showed positive reactions at both bovine and avian tuberculin injection sites and a strong association (χ 2 = 2.512; P = 0.113) between skin responses to both tuberculins. The study confirms that bovine TB is prevalent in live cattle and meat production abattoirs in Cameroon and we recommend strict a interpretation of TST results for maximum diagnosis of the disease in the local environment. A need for comprehensive investigation of the molecular epidemiology, zoonotic risks and the public health importance of bovine TB in Cameroon cannot be overemphasised.