Long‐whiskered catfish, Sperata aor (Hamilton 1822), is commercially important in food, ornamental and sport fisheries. The fish is mainly caught from the wild populations because its aquaculture practices are not commercialised. Inland fishery in the Ganga basin is mostly unorganised; hence, no published report is available on the trend of S. aor production from the selected habitat. In India, S. aor has been categorised vulnerable mainly due to natural and anthropogenic threats. Otolith chemistry shows variation with changing physico‐chemical conditions of the fish habitat. Therefore, the present study was conducted with the objective to analyse spatio‐temporal variations in water chemistry in relation to environmental factors; relationship between water and otolith chemistry; and spatio‐temporal variations in otolith chemistry to discriminate the stocks of S. aor inhabiting the River Ganga. Most of the element: Ca ratios in water samples did not show significant correlations with environmental factors, viz. temperature and conductivity. Only few element: Ca concentrations in otoliths were positively correlated to their corresponding ratios in the ambient water. In the selected study area, the S. aor populations were discriminated into four stocks possibly because of heterogeneous water chemistry at the sampling sites, and physical barriers. In the present study, otolith chemistry showed relatively low temporal variability as compared to spatial variability; thus, the classification accuracy of individuals to their original populations remained consistent over the selected time period. The findings could be useful in devising scientifically sound management strategies and/or any conservation plans for the vulnerable S. aor populations inhabiting the River Ganga.