In this study, tobacco leaf samples (n = 26) were collected from different areas in Kushtia to determine the presence of residues of cypermethrin, diazinon, heptachlor, methoxychlor dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethan (DDD) and dichlorodiphenyldichloroethylene (DDE). The analysis was conducted by a High Performance Liquid Chromatography (HPLC) system that was equipped with a photodiode array detector. Both cypermethrin and diazinon were detected in tobacco samples from six districts, namely, Mirzapur, Shahebnagar, Kodalipara, Pragpur, Farakpur and Taragunia. The highest concentration of cypermethrin was found in Kodalipara (2.00 ppm) while the highest concentration of diazinon was detected in a sample from Pragpur (0.15 ppm). The pesticide DDT was only detected in the sample from Pragpur at 4.00 ppm. This is the first study in Bangladesh that reports pesticide residue concentrations in tobacco leaf samples.