Mycobacterium tuberculosis complex are important pathogens to man and causative agents of tuberculosis. Tuberculosis is one of the diseases that continue to be a public health concern in the world. Earlier, tuberculosis was considered controlled, but with emergence of HIV and AIDS, the disease has been exacerbated, with the highest incidences being in subSaharan Africa. Kenya ranks 15th and 5th in the World and Africa respectively. The World Health Organization indicates that there were 300 TB cases per 100,000 people in 2011. Information on the identity of the Mycobacterium tuberculosis complex and the prevalence of pulmonary tuberculosis in HIV positive patients seeking treatment in Kisumu County is limited. In this study, we isolated and identified the Mycobacterium tuberculosis complex and also determined the prevalence of tuberculosis in HIV patients seeking treatment in Kisumu County. A cross-sectional study was conducted between December 2013 and June 2014. The study engaged, 379 HIV positive patients suspected of TB infection who gave sputum samples. The sputum samples were then decontaminated, concentrated, liquefied and neutralized before being cultured in liquid media using MGIT 960 tubes. The culture positive MGIT tubes were sub cultured in Brain Heart Infusion Agar (BHIA) before microscopic examination of the culture using ZN smear for Acid Fast Bacilli and identified using Genotype MTBC. In the study, 130 (34.3%) of the 379 suspected TB patients were diagnosed positive for pulmonary TB by MGIT culture. A significantly greater number of males were diagnosed with pulmonary tuberculosis compared to females (57.7% and 42.3% respectively; χ2 = 1.0342, df = 1 P < 0.05). Further, the study revealed that TB prevalence decreased steadily with age, with the youths being at greater risk of becoming active patients. In the age category of 26-35 years, prevalence was 36.92% while, for age category above 55 years it was 6.15%. Based on the results, the etiological agent of tuberculosis in Kisumu County is Mycobacterium tuberculosis, the prevalence of infection was higher in males than in females and the prevalence rate to infection was highest among the youth. There is need for rapid and accurate diagnosis of active TB particularly in HIV-positive patients. Also, TB awareness and control programme should mainly be directed towards the youths by the all the stakeholders in the Public Health sector.