Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), is a cosmopolitan and destructive pest in rice fields of the world. This pest was reported in 1973 in Iran, and it has since spread widely in rice, Oryza sativa L., fields throughout the country. In this study, we tried to evaluate comparative toxicity of diazinon in five colonies of C. suppressalis, collected from Babol (Ba), Amol (Am) of Mazandaran Province and Rasht (Ra), Sheikhmahale (Sh), and Gourabzarmikh (Go) of Guilan Province, northern Iran. The LD₅₀ values were compared. We also evaluated the general esterases, alkaline phosphatase (ALP), glutathione transferase (GST), and acetylcholinesterase (AChE) activities from the five populations. The LD₅₀ values of Ra, Ba, Am, and Sh (12.64, 11.4, 7.17, and 3.71 µg/ mg larva-1) were 13.67-, 12.33-, 7.75-, and 4.02-fold higher than Go population (0.924 µg/mg larva-1). Using α-naphthyl acetate as substrate, the general esterase activities in Ra, Ba, Am, and Sh colonies were, respectively, 1.81-, 1.68-, 1.75-, and 1.35-fold more than those in Go population. When β-naphthyl acetate was used as the substrate, activity ratio was measured 1.98-, 2.58-, 1.25-, and 1.24-fold compared with the Go population. Glutathione transferase activities in Ra, Ba, Am, and Sh populations were 1.27-, 1.68-, 0.98-, and 1.7-fold more than those in Go, when 1-chloro-2,4-dinitrobenzene was used as the substrate. When 1,2-dichloro-4-nitro-benzene was used as the substrate, activity ratio was measured 1.14-, 1.42-, 0.56-, and 0.95-fold compared with Go population. The ALP activity demonstrated a significant difference among these populations and in Ra, Ba, Am, and Sh larvae were 3.54-, 4.62-, 3.84-, and 2.18-fold more than Go. The AChE inhibition or I50 value was 0.19, 0.22, 0.31, 0.19, and 0.26 mM in Ra, Ba, Am, Sh and Go populations, respectively. However, the results showed no significant differences in studied colonies. These biochemical characterizations of general esterases ALP, GST, and AChE were consistent with diazinon bioassay in the five populations. It is inferred from increased esterase, alkaline phosphatase and glutathione transferase, activities that might play an important role in the increasing resistance in C. suppressalis to diazinon among these five populations.