This study contributed to understanding the variation in morphometrics of Mystus mysticetus. In this study, 451 individuals were collected in two ecological regions, including the brackish region in Long Phu, Soc Trang, and the freshwater region in Cai Rang, Can Tho, Vietnam. Fish samples were collected continuously for nine months, from October 2020 to June 2021. Data analysis results showed that sex, season, and site variables regulated the variations of fish total length (TL) and weight (W). These two parameters were also affected by sex × season, sex × site, and season × site. Morphometrics of this fish, such as eye diameter (ED), eye distance (DE), body high (BH), head length (HL), HL/TL, BH/TL, DE/HL, and ED/HL fluctuated with the season, site but not sex. These parameters of the species were not affected by sex × season and sex × site but varied with season × site. The findings contribute to fish identification and support ecological adaptation measures in the studied regions.