Carotenoid content in durian (Durio zibethinus) fruit is an important aspect of fruit quality. We analyzed fruit size, color, carotenoid composition and expression of genes involved in carotenogenesis of two durian cultivars ‘Chanee’ and ‘Monthong’ with differences in pigmentation during growth and ripening. The ripe fruit of ‘Monthong’ has a greater diameter and fruit weight than that of ‘Chanee’, while total carotenoid concentration was approximately 6-fold higher in ‘Chanee’ than in ‘Monthong’. The major carotenoids in the fruit pulp were β-carotene and α-carotene, with minor carotenoids being lutein and zeaxanthin. Significant differences in α-carotene and β-carotene levels were seen between the two cultivars. We cloned fragments of PSY, PDS, ZDS, CRTISO, LCYB, LCYE, CYCB, BCH, ECH and Or genes from durian. Expression analysis of the carotenoid biosynthetic genes using qPCR showed that ZDS expression positively correlated with increases in total carotenoids, β-carotene, α-carotene, and lutein content and LCYE expression correlated with total carotenoids and β-carotene content. In contrast, LCYB showed a negative correlation with total carotenoids, β-carotene, α-carotene and lutein. It can be concluded that carotenoid accumulation during durian fruit growth is regulated by the balanced expression of the ZDS, LCYE and LCYB genes.