The aim of this work was to evaluate the carotenoid content and genetic variability of banana accessions from the Musa germplasm collection held at Embrapa Cassava and Tropical Fruits, Brazil. Forty-two samples were analyzed, including 21 diploids, 19 triploids and two tetraploids. The carotenoid content was analyzed spectrophotometrically and genetic variability was estimated using 653 DArT markers. The average carotenoid content was 4.73 μg.g -1 , and ranged from 1.06 μg.g -1 for the triploid Nanica (Cavendish group) to 19.24 μg.g -1 for the triploid Saney. The diploids Modok Gier and NBA-14 and the triploid Saney had a carotenoid content that was, respectively, 7-fold, 6-fold and 9-fold greater than that of cultivars from the Cavendish group (2.19 μg.g -1). The mean similarity among the 42 accessions was 0.63 (range: 0.24 to 1.00). DArT analysis revealed extensive genetic variability in accessions from the Embrapa Musa germplasm bank.