The aim of this study was to characterize 160 clinical Mycobacterium tuberculosis isolates from Guangdong with respect to their drug susceptibility phenotypes to three common anti-tuberculosis drugs, isoniazid (INH), rifampin (RIF) and streptomycin (SM), and with respect to genetic mutations in the most commonly corresponding resistance genes (katG, rpoB and rpsL). The drug susceptibility profiles were evaluated by the absolute concentration method, and genetic mutations in the corresponding resistance genes were identified by DNA sequencing. Among these isolates, 33.1% (53/160) were drug-resistant. The percentages of isolates resistant to INH, RIF and SM were 21.9% (35/160), 16.9% (27/160) and 15.6% (25/160), respectively. Twenty-five of 35 (71%) INH-resistant isolates, 22 of 27 (81.5%) RIF-resistant isolates and 19 of 25 (76%) SM-resistant isolates were found to have mutations in the analyzed katG, rpoB and rpsL gene fragments. The most frequent mutation patterns for the three drugs were as follows: INH, Ser315Thr (68.6%) in katG; RIF, Ser531Leu (55.6%) in rpoB; and SM, Lys43Arg (72%) in rpsL. These findings provide useful data on the mutation types of drug-resistant genes in M. tuberculosis isolates from Guangdong province in China.