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• The new allelic variant found in CsaV3_7G029170 (CsaYf) linked to yellow endocarp in the PI200815 (CS-C) line via high-throughput sequencing. • A missense mutation in CsaYf changed the original Aspartic acid (D) in the JEF, KWSA, and CS-A (MEJ) line to Asparagine (N) in the CS-C line. • Concordant phenotype and genotype results suggest a 3:1 ratio and denote a single recessive gene inheritance for the yellow fruit flesh trait. • Downregulation of the CsaYf gene is inversely correlated with ß-carotene accumulation in the CS-C line's meso and endo carp tissues, indicating a need for further investigation. Cucumber (Cucumis sativus) is a globally valued and widely cultivated vegetable crop. However, most commercially available cucumbers contain white or green fruit flesh color, which has very low ß-carotene and other carotenoids. Conversely, orange or yellow-fleshed cucumbers are rich in carotenoids and offer significant dietary and nutritional benefits. To generate cucumber varieties with carotenoid-enriched fruit flesh, we conducted high-throughput sequencing of a yellow-fleshed cucumber PI200815 (CS-C) and white-fleshed cucumbers JEF, and KWSA, to identify the genomic region responsible for yellow-fleshed fruit. Building on the previous report by Lu et al. (2015), we identified seven unique DNA variations in five genes specific to the CS-C line using HiSeq2000 and genomic mapping to 'Chinese-Long v3′. Further, based on the Sanger gene sequencing, among the five candidate genes, we found the CsaV3_7G029170 (encodes for an ATP-binding cassette (ABC) transporter C family member 4 [ CsaYf ]) gene with a single nucleotide polymorphism (SNP) variation in the third exon resulted in a missense mutation caused an amino acid change from Aspartic acid (Asp) to Aspargine (Asn) in the CS-C line. Accordingly, the gene segregation analysis based on the SNP variation revealed that the CsaYf gene confers the yellow endocarp trait in the CS-C line through recessive Mendelian single-gene inheritance. Notably, we observed a negative correlation between the downregulation of CsaYf gene expression and the accumulation of ß-carotene in meso and endo carp tissues in the CS-C line, suggesting a further investigation into the underlying molecular mechanism. This finding provides a novel genetic basis for breeding cucumbers with enriched carotenoid content to enhance their nutritional value. [ABSTRACT FROM AUTHOR]