Abstract Fishes are excellent subjects for the study of the mutagenic and carcinogenic potential of contaminants present in water. Herbicides are one of the toxic contaminants that cause lethal effects in fish. In this context, the present study was designed to study the sub‐lethal toxic effect (low dose [LD] 25 mg/L, medium dose‐50 mg/L, and high dose [HD] 100 mg/L) of a commonly used herbicide, pyrazosulfuron ethyl (PE), on erythrocytes of tilapia. The genotoxic effect was studied to understand the sub‐lethal potential by means of (a) the nuclear abnormalities, including the micronucleus (MN) frequency; (b) further, each stage of the cell cycle was analysed using flow cytometry and (c) finally, the targets of PE was deciphered using in silico analysis. The results showed that there were significant effects of time (7 and 14 days) and doses in the increase in the frequency of MN in erythrocytes of fish treated with PE, compared to the control group. After exposing fish to PE for 7 and 14 days, there was a proportional increase in the frequency of the apoptotic population of erythrocytes for LD to HD. During both time exposures (7 and 14 days), the highest cell death was accounted for in HD. Moreover, the majority of the cells showed a dysregulated cell cycle in the G1 phase in all the treated groups of PE, compared to the control group. This study showed that PE might initiate genotoxicity in non‐target species (tilapia) of the aquatic ecosystem near agricultural fields.