DNA damage of peripheral lymphocytes in 60 workers occupationally exposed to trivalent chromium [Cr(III)] in a tannery was studied using comet assay. The urinary and blood chromium levels were detected as a biomarker of internal exposure. The 90 subjects were divided into three groups: (i) exposure group I included 30 tannery workers highly exposed to chromium from tanning department; (ii) exposure group II included 30 tannery workers with moderate chromium exposure from finishing department; (iii) control group included 30 individuals without exposure to physical or chemical genotoxic agents. No significant difference was found among the three groups for age and smoking. The results showed that the medians of blood and urinary Cr of two exposure groups were significantly higher than those of control group (P<0.01). And the medians of blood and urinary Cr of exposure group I were significantly higher than those of exposure group II (P<0.05 or P<0.01). The medians of mean tail length (MTL) of the three groups were 5.33 (2.90-8.50), 3.43 (2.31-8.29) and 2.04 (0.09-3.83) microm, respectively; The medians of mean tail moment (MTM) of the three groups were 6.28 (2.14-11.81), 3.41 (1.25-11.07) and 0.53 (0.13-3.29), respectively. The MTL and MTM of two exposure groups were significantly higher than those of control group (P<0.01). The MTL and MTM of exposure group I were significantly higher than those of exposure group II (P<0.01). The results of the present investigation suggest that occupational exposure to trivalent chromium can lead to a detectable DNA damage of human peripheral lymphocytes. Moreover, DNA damage was associated with chromium levels in blood. DNA damage may serve as a valuable effective biomarker and total chromium in blood may serve as a useful internal exposure biomarker in the population occupationally exposed to trivalent chromium.