In the present study, carotenoid concentrations and profiles of carotenoids were determined at harvest and during storage in fruit of several cultigens of winter squash, including several New Hampshire (NH) breeding lines, the cultivars Waltham Butternut and Puritan Butternut in Cucurbita moschata (Duch.), and the hybrid cultivars Sunshine, Eclipse, Space Station, and Thunder in Cucurbita maxima (Duch.). Cultigens were grown at research farms in either Madbury or Durham, NH, in 2007, 2008, and 2009, and fruit were harvested at either 40 or 60 days after pollination (DAP), and stored for 0, 30, or 60 days at 14 °C before sampling. Total carotenoid concentrations were determined spectrophotometrically and carotenoid profiles were determined using high-performance liquid chromatography (HPLC). Carotenoid concentrations in fruit samples of C. maxima cultigens harvested at 60 DAP ranged from 146 to 320 μg·g−1 fresh weight (FW), compared with concentrations of 274 to 623 μg·g−1 FW after storage for 60 days. Carotenoid concentrations were lower among C. moschata cultigens, ranging from 42 to 145 μg·g−1 FW at 60 DAP, and from 84 to 239 μg·g−1 FW after 60-day storage. The nutritionally important carotenoids, lutein, zeaxanthin, and β-carotene, comprised 41% to 63% of the total carotenoid profile in the C. maxima kabocha hybrids; whereas, the carotenoids neoxanthin and flavoxanthin comprised 37% to 59%. β-Carotene and lutein were the major nutritionally beneficial carotenoids identified in ‘Waltham Butternut’ and three inbred lines of C. moschata, along with much smaller concentrations of α-carotene. Neoxanthin and violaxanthin comprised between 14% and 29% of the total carotenoid profiles among the four cultigens analyzed. In the popular cultivar Waltham Butternut, the β-carotene concentration was 17.3 μg·g−1 FW at 60 DAP, but increased to a maximum of 57.8 μg·g−1 FW after storage for 30 days; whereas, lutein concentrations increased from 27.3 μg·g−1 FW at 60 DAP to 44.6 μg·g−1 FW after 60-day storage. In Sunshine, a popular kabocha cultivar, β-carotene increased from 26.0 μg·g−1 FW at 60 DAP to 104 μg·g−1 FW after 60-day storage. Lutein + zeaxanthin concentrations in ‘Sunshine’ were 58.8 μg·g−1 FW at 60 DAP, but increased only to 71.8 μg·g−1 FW after 60-day storage. Total carotenoid concentrations and carotenoid profiles in ‘Sunshine’ over three growing seasons, and in ‘Waltham Butternut’, ‘NH.Mo421’, and ‘NH.Mo851’ over two growing seasons, were similar.