Hilsa, Tenualosa ilisha is a commercially important and high-valued anadromous fish of South East Asian countries. The present study aimed to understand macro and microscopic changes in the ovary and mobilization of fatty acids during gonad development in wild-caught female hilsa. A total of 174 female fish samples of different size groups (Weight range: 68.20 ± 3.21 to 460.67 ± 29.89 g, Total length range: 18.53 ± 0.55 to 40.33 ± 1.20 cm) were collected from estuarine and freshwater zones of the Hooghly River system of India. Various biological indices, such as gonadosomatic index (GSI), hepatosomatic index (HSI), oocyte diameter (OD), viscerosomatic index (VSI) and condition factor (K) at different maturation stages (I to VI) were determined. Results of the study showed that the GSI and OD increased with ovarian growth from stage I to VI (14.50 ± 2.18%) of maturation (P < 0.05). The HSI value increased progressively from stage I to V; thereafter, it decreased (P < 0.05). Histological observations indicated different microscopic stages, namely oogonia (stage I), chromatin nucleolar oocytes, perinuclolar oocytes (stage II), cortical alveolar (III), vitellogenic (IV), post-vitellogenic (V) and hydrated/eccentric stages (VI). Total saturated fatty acid (∑SFA) decreased in the ovary and increased in the liver with the progress of maturation from stage I to VI. At the same time, total monounsaturated fatty acid (∑MUFA) in muscle remained unchanged (P > 0.05), decreased (P < 0.05) in the liver, and increased in the ovarian tissue (P < 0.05). The levels of n-3 PUFA (polyunsaturated fatty acid) and n-6 PUFA elevated in the ovarian tissue concomitantly with the ovarian growth (P < 0.05). The ratios of n-3/n-6, docosahexaenoic acid (DHA) / eicosapentaenoic acid (EPA), EPA/ arachidonic acid (AA) and DHA/AA showed a significant decreasing trend in the ovarian tissue with the progress of gonad development (P < 0.05). Accumulation of n-3 PUFAs (α-linolenic acid, ALA; EPA; DHA) and n-6 PUFAs (linoleic acid, LA and AA) in ovary suggested specific fatty acid requirements during gonad development, which will help in the formulation of a broodstock diet for hilsa.