Abstract: Red palm oil has high nutritional value due to its richness in carotene, so the separation and detection of carotene isomers in red palm oil are of practical value. In the paper, high-performance liquid chromatographic separation of four carotene isomers (α-carotene, all-trans-β-carotene, 13-cis-β-carotene, 9-cis-β-carotene) in red palm oil was optimized in detail. Within three chromatographic columns including InertSustain C18, Welch Ultimate XB-C30 and YMC C30, the effects of mobile phase type, elution mode, mobile phase ratio, flow rate, and column temperature on the separation of four target analytes were studied. The experimental results showed that the type and ratio of mobile phase, especially the type of chromatographic column and column temperature, have a great influence on the chromatographic separation performance of carotene isomers in red palm oil. The optimized chromatographic conditions were:YMC C30 as chromatographic column, dichloromethane/acetonitrile/methanol (20.5:15:64.5, V/V/V) as mobile phase, isocratic elution mode, 1.2mL/min of flow rate, 25℃ of column temperature. Under the optimized chromatographic conditions, four carotene isomers in red palm oil were successfully separated and the analytical features of proposed method were evaluated. The method showed a good linear relationship in the range of 0.2-3.0μg/mL with a correlation coefficient (R²) ranging between 0.9996 and 0.9999. The limit of detection, defined as a signal-to-noise ratio (S/N) of 3, was 0.01μg/mL. According to the oil sample treatment method in GB 5009. 83-2016, the content of four carotene isomers in palm oil was determined under the optimized chromatographic separation condition after ethanol extraction and saponification. Experimental results showed that the content of 13-cis-β-carotene, α-carotene, all-trans-β-carotene, and 9-cis-β-carotene in red palm oil was 16. 9,40.2,42.9, and 5.1mg/kg, respectively. The spiked recoveries of four carotene isomers in red palm oil were 91.0%-116.0%, with the relative standard deviations (RSDs) from 2.3% to 8.5% (n=5). The results indicated that the proposed method could be successfully used for the separation and detection of four carotene isomers in red palm oil, and had great application potential in the quality analysis of red palm oil. However, during the test, the structure of carotene was unstable, easily degraded by heat, light and oxygen, and the actual oil sample matrix was complicated, and these problems should be paid great attention in the process of extraction and preservation.