Carotenoids are pigments responsible for the yellow-reddish color of many foods and are related to important functions and physiological actions, preventing several chronic-degenerative diseases. The objective of this study was to confirm the carotenoid composition of jackfruit by high-performance liquid chromatography connected to photodiode array and mass spectrometry detectors (HPLC-PDA-MS/MS). The main carotenoids were all-trans-lutein (24–44%), all-trans-β-carotene (24–30%), all-trans-neoxanthin (4–19%), 9-cis-neoxanthin (4–9%) and 9-cis-violaxanthin (4–10%). Either qualitative or quantitative differences, mainly related to the lutein proportion, were found among three batches of jackfruit. Since the fruits from batch A showed significantly lower contents for almost all carotenoids, it also had the lowest total carotenoid content (34.1 μg/100 g) and provitamin A value, whereas the total carotenoid ranged from 129.0 to 150.3 μg/100 g in the other batches. The provitamin A values from batches B and C were 3.3 and 4.3 μg RAE/100 g, respectively. The carotenoid composition of jackfruit was successfully determined, where 14 of the 18 identified carotenoids were reported for first time. Differences among batches may be due to genetic and/or agricultural factors.