We developed easy, non-destructive methods to investigate the presence of carotenoid and flavonoids in petals by observing them under blue and ultraviolet lights, which are characteristically absorbed by yellow pigments. By using these methods we identified a Eustoma cultivar whose yellow petal coloration was attributed to its carotenoid content. The identification refutes the notion that yellow Eustoma cultivars lack carotenoids. Hence, deep yellow cultivars of this species may be bred in the future. Carotenoids in yellow petals of Eustoma were identified as all-E-neoxanthin, 9′-Z-neoxanthin, violaxanthin, lutein, zeaxanthin and β-carotene. Being different in green bud, most carotenoids occurred as conjugates in open petal, which suggests a stage-specific regulation of carotenoid metabolism. The qualitative and quantitative methods used in this report could be applied to identify other carotenoid-derived yellow flower cultivars in other horticultural species.