Potential mutagenic and genotoxic effects of Chlorthiophos, an organophosphate pesticide, were evaluated using four standard assays. Five different concentrations of the pesticide were tested by an Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102, with and without S9 metabolic activation. No concentrations of Chlorthiophos showed mutagenic activity on the TA97, TA100, and TA102 strains, with and without S9 fraction, but were all mutagenic to the TA98 strain without S9. Sister chromatid exchange (SCE), chromosome aberration (CA), and micronucleus (MN) tests were used to investigate the genotoxic effects of Chlorthiophos in human peripheral lymphocytes treated with 25, 50, 100, and 200 µg/mL concentrations of Chlorthiophos for 24 and 48 h. The nuclear division index (NDI), replication index (RI), and mitotic index (MI) were also calculated to determine the cytotoxicity of Chlorthiophos. No increase in SCE frequency was seen for any treatment period or concentration, but Chlorthiophos at 200 µg/mL increased the frequency of CAs. Increases in MN formation were only observed at Chlorthiophos concentrations of 200 µg/mL following 24 and 48 h treatments. Chlorthiophos treatment reduced the MI and NDI significantly, but had no effect on the RI. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 937–945, 2015.