Using stable isotope analysis, this study examined the potential food sources of the Japanese scallop, Patinopecten (Mizuhopecten) yessoensis (Jay) and determined whether the isotopic ratios inferred from tissues of cultured scallops varied according to culture systems (hanging and sowing). Different tissues (i.e., muscle, ctenidia, and digestive gland) of scallops nesting in Saloma Lake (18.3–113.0 mm shell height) and Tokoro (66.8–121.4 mm shell height) and their potential food sources were analyzed for stable carbon and nitrogen isotope ratios. Average isotope ratios obtained in scallop muscle and ctenidia (−20.6 and −21.0‰ for δ¹³C; 3.3 and 3.0‰ for δ¹⁵N) from Tokoro area (sowing culture; ∼65 m depth) had close affinities to suspended particulate organic matter (−21.6‰ and 2.7‰ for δ¹³C and δ¹⁵N, respectively) suggesting that the scallops from Tokoro area depends on detritus as an important food source. Saloma Lake (hanging culture; ∼20 m depth) scallop muscle and ctenidia tissues however had relatively enriched δ¹³C values (−17.7 and −18.1‰) and were relatively closer to either net plankton (−21.1‰) and particulate organic matter (−22.1‰). δ¹⁵N values were also higher in particulate organic matter taken in Saloma Lake (5.0‰) and showed a 5.1‰ difference when muscle tissues in two sites are compared. Digestive gland tissues however showed depleted δ¹³C and δ¹⁵N signatures relative to other tissues in both systems. The observed variations in isotopic ratios in scallops reared in two systems thus reflect differences in carbon and nitrogen sources related to culture habitats.