Photosynthesis serves as the primary source of nutrients synthesized in higher plants, and improving photosynthetic efficiency can significantly increase crop yield and fruit quality. Leaf color mutants represent ideal materials for studying chloroplast development and photosynthesis mechanisms and have been widely characterized in field crops. However, relevant research on watermelon leaf color mutants remains scarce. In this study, we isolated a yellow-green phenotype mutant, PKH352, from an EMS-mutagenized watermelon mutant library. The chlorophyll content and maximal photochemical efficiency in PKH352 were significantly decreased. Genetic analysis showed that the mutated trait was controlled by a single nuclear gene, which was named Clygp (Citrullus lanatus yellow-green plant). Through MutMap and linkage analysis in an F₂ population of 440 plants, we identified a single nucleotide polymorphism (SNP) mutation within ClG42_04g0106300, which encoded a signal recognition particle 54 kDa protein, as the causal variant for the yellow-green phenotype. Further validation using a CRISPR/Cas9-mediated system confirmed that knockout of ClG42_04g0106300 results in the yellow-green phenotype in watermelon. In addition, comparative transcriptomic analysis revealed that mutations in ClG42_04g0106300 greatly affected the expression of key genes associated with chloroplast development and photosynthesis, providing strong evidence that this gene plays a critical role in these biological pathways. Taken together, these findings provide insights into the molecular mechanisms underlying chloroplast development and photosynthetic efficiency, offering a theoretical basis for breeding watermelon varieties with high photosynthetic efficiency.