This study determined vitamin D content in commonly consumed fish in Thailand and the effects of different cooking methods on vitamin D retention. Five species of freshwater fish and four species of marine fish were purchased from three representative markets. All of the fish were individually prepared according to common household practices. Vitamin D2 and D3 were determined using the HPLC standard method (AOAC method 995.05). The results indicated that vitamin D3 was the only detectable form of vitamin D in the fish. Vitamin D content of raw freshwater fish ranged from 2.42 to 48.5 µg per 100 g edible portion (EP), which was higher than that of raw marine fish (2.94 to 4.69 µg per 100 g EP). Common silver barb, Red Nile tilapia, and Nile tilapia (freshwater fish living in the limnetic zone) contained high levels of vitamin D (48.5 ± 26.5, 31.0 ± 7.7, and 19.8 ± 3.5 µg per 100 g EP, respectively). Boiled fish (except for Common silver barb), fried fish (except for Striped snakehead, Walking catfish, and Common silver barb), and grilled fish (except for Common silver barb, Giant sea perch, and Short-bodied mackerel) retained high levels of vitamin D, which were not significantly different (p > 0.05) from raw fish. Common silver barb, Red Nile tilapia, and Nile tilapia—cooked by boiling, frying, and grilling—are recommended for consumption as excellent sources of vitamin D.