[Objective] To elucidate the relative polarity and stability of seven carotenoids accumulated in normal spirilloxanthin biosynthesis pathway of anoxygenic phototrophic bacteria (APB) by thin-layer chromatography (TLC). [Methods] We investigated the changes in polarity, characteristic absorption spectra and stability of seven carotenoids accumulated in spirilloxanthin biosynthetic pathway of Rhodopesudomonnas palustris CQV97 by TLC, image gray intensity analysis, absorption spectroscopy and HPLC analysis. [Results] Strain CQV97 accumulated seven types of carotenoids including lycopene (C1), rhodopin (C2), 3,4-didehydrorhodopin (C3), anhydrorhodovibrin (C4), rhodovibrin (C5), OH-spirilloxanthin (C6) and spirilloxanthin (C7), the seven carotenoids were clearly separated by TLC. The relative polarity of seven carotenoids on TLC plate changed in the order of C1C3 ≈C7>C5≈C4≈C1≈C6. In darkness, the seven carotenoids were stable within 90 min. [Conclusion] TLC was capable of resolving clearly all seven carotenoids accumulate in spirilloxanthin biosynthetic pathway of APB. The seven carotenoids on TLC plate were sensitive to illumination and short-term stable in darkness. The results will be helpful for rapid identification of carotenoids by TLC.