Mycobacterium tuberculosis strains collected from patients with pulmonary tuberculosis in the West and Centre regions of Cameroon were culture-tested for the major anti-tuberculosis drugs (isoniazid, rifampicin, ethambutol and streptomycin). Of the 112 predetermined samples included in the study, 21 (18.7%) were sensitive to all the drugs, while 91 (81.3%) were resistant to at least one drug. Resistance to isoniazid was the most common (79.1%), followed by rifampicin (65.9%), streptomycin (62.6%) and ethambutol (38.5%), 50% of the samples were qualified as (resistant to at least isoniazid and rifampicin). A PCR-based dot-blot hybridization strategy was used to detect mutations at different loci in five genes associated with resistance to the drugs tested. For rifampicin resistance, the mutation on codon 526 of the rpoB gene was the most common (66.3%), followed by the codon 516 (60.5%) and the codon 531 (31.4%). The mutation on codon 513 of the rrs gene was the most encountered in streptomycin resistant strains (77.8%); while the mutation on codon 43 of rpsL gene was always associated to that of rrs gene. The mutation on codon 531 of the rpoB gene for rifampicin resistance (95.6%) was most prevalent in the samples from the Centre region compared to the West region (P = 0.0003). Generally, no significant differences were obtained on the prevalences of the other mutations analysed based on the regions, gender or the age of patients (P > 0.05). The dot-blot analysis, gave no result for the codon 306 of embB gene associated with ethambutol resistance under experimental conditions used in this study. The PCR-based dot-blot hybridization strategy was tested to validate the procedure on stored samples and could be a good surveillance method for rapid detection of the evolution of drug resistant M. tuberculosis in Cameroon.