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The thraustochytrids, the heterotrophic marine microorganisms, have received increasing attention because of their capacity for the production of high‐value products such as docosahexaenoic acid (DHA), carotenoids, and sterols. In this study, three potential Δ5,7‐sterols, showing similar UV absorption spectra to that of ergosterol, in the thraustochytrid Schizochytrium sp. S31, were estimated by the ultraperformance liquid chromatography (UPLC) − APCI−MS/MS method. The proposed structures of three Δ5,7‐sterols were ergosta‐5,7,24(28)‐trien‐3β‐ol, ergosta‐5,7‐dien‐3β‐ol, and 24‐ethylcholesta‐5,7,22‐trien‐3β‐ol. The contents of astaxanthin, ergosterol, and the other three Δ5,7‐sterols in Schizochytrium sp. S31 during the cultivation performed in three separate batches were analyzed by a fast UPLC–PDA method. The results demonstrated that astaxanthin, ergosterol, and ergosta‐5,7‐dien‐3β‐ol were the predominant carotenoid and Δ5,7‐sterols, respectively, and their contents kept increasing during the growth and reached a maximum after 23 days of cultivation. The estimated maximum contents of astaxanthin and four Δ5,7‐sterols, including ergosterol, ergosta‐5,7‐dien‐3β‐ol, ergosta‐5,7,24(28)‐trien‐3β‐ol, and 24‐ethylcholesta‐5,7,22‐trien‐3β‐ol were 2.31, 4.02, 3.43, 0.30, and 0.97 mg g−1 dry biomass, respectively. These findings are different to other strains reported in the composition and accumulation of Δ5,7‐sterols and carotenoids. The present study suggests that Schizochytrium sp. S31 might be a promising marine microorganism for the productions of high‐value natural pigment astaxanthin, the provitamin D2 ergosterol, and the provitamin D4 ergosta‐5,7‐dien‐3β‐ol, and taken as a model organism to study the sterologenesis of the microorganisms because of its complexity of sterol compositions. [ABSTRACT FROM AUTHOR]