Stem color is an important agronomic trait in zucchini ( Cucurbita pepo , C. pepo ) for robust seedings and high yield. However, the gene controlling this trait has not been characterized. This study identified a single locus accounting for dark green stem color in C. pepo ( CpDsc-1 ). Genetic analysis of this trait in segregation populations derived from two parental lines (line 296 with dark green stem and line 274 with light green stem) revealed that it was controlled by a single dominant gene (dark green vs. light green). Using the bulked segregant analysis method, CpDsc-1 was mapped to a 2.09 Mb interval on chromosome 15. Then this region was further narrowed down to 65.2 kb using linkage analysis in the F 2 population. Sequencing analysis revealed a 14 kb deletion between Cp4.1LG15g03420 and Cp4.1LG15g03360 , encoding a two-component response regulator-like protein (APRR2). The incomplete structures of the two APRR2 genes and the abnormal chloroplasts in line 274 might be the main reason for the light green phenotype. Gene expression pattern analysis showed that only Cp4.1LG15g03420 was upregulated in line 296. The subcellular localization analysis indicated that Cp4.1LG15g03420 was a nuclear gene. Furthermore, a co-dominant marker G4563 (93% accuracy rate) and a co-segregation marker Fra3 were established in 111 diverse germplasms, and both were tightly linked with the color trait. This study provide new insight into the chlorophyll regulation pathway, and these markers would be valuable for marker-assisted selection in zucchini breeding.