The study conducted an investigation into the reproductive biology of M. pancalus and assessed the influence of water quality parameters and environmental factors on the spawning pattern within the Gajner Beel ecosystem in Bangladesh. A total of 1218 individuals of M. pancalus (46.39% males and 53.61% females) were collected monthly from the Gajner Beel during January to December 2018 using various fishing gears. The total length (TL) of each individual was measured using digital slide calipers, and the whole body weight (BW) was measured using an electronic balance. Fulton's conditions factor (KF) showed significant differences between males and females. The calculated Lm were 11.11 cm, 11.30 cm, and 11.10 cm based on maximum length, gonadosomatic index (GSI), and the logistic model. The spawning season extended from May through August, with June and July being peak months. The average total fecundity was 1495.52 ± 840.24, with a range of 370 to 4069. During peak spawning season, the average temperature and rainfall were 27°C and 370 mm, respectively. Rainfall, dissolved oxygen, total alkalinity, and pH all had a significant (p < 0.01) positive effect whereas temperature and TDS all had a significant (p > 0.01) negative effect on GSI. Annual air temperature in the study area increased by 0.053 °C/year, with a regression coefficient value (r2 = 0.1695), while annual mean rainfall decreased by 5.97mm/year (r2 = 0.076). This research will contribute to the development of conservation and management approaches of Mastacembelidae fish in relation to current climate variability in sub-tropical waters.