Chloroplasts drive photosynthesis and regulate cellular signaling and phytohormone biosynthesis processes that greatly influence crop yield and quality traits. In this study, we identified a recessive chlorophyll-deficient mutant, reduced chlorophyll mutant 1t (rcm1t), from an advanced-generation tomato inbred line. Genetic and phenotypic characterization confirmed that chloroplast degradation accelerates rapidly and chlorosis occurs during late leaf development in the rcm1t mutant. Subsequently, we fine-mapped rcm1t to an approximately 60 kb region on chromosome 8. This interval encompasses Lutescent (L1)/SlRCM1, a well-characterized gene essential for chloroplast biogenesis and developmental maintenance. An allelism test between rcm1t and the previously characterized ethyl methanesulfonate (EMS)-induced rcm1 mutant revealed non-complementation. Moreover, transgenic complementation with the wild-type SlRCM1 coding sequence rescued the chloroplast premature senescence phenotype in rcm1t, providing definitive genetic evidence that rcm1t is a loss-of-function allele of SlRCM1. Resequencing identified a ~4.8 kb retrotransposon insertion in the first exon of the gene, which disrupts normal transcription and results in a premature stop codon. Quantification of abscisic acid (ABA) revealed significantly higher ABA levels in the rcm1t mutant than in the wild type. Protein–protein interaction analyses demonstrate that SlRCM1 interacts with SlNCED2. Osmotic stress assays showed that the rcm1t near-isogenic line exhibited enhanced tolerance under osmotic stress. This study successfully mapped the natural allele rcm1t of SlRCM1, preliminarily explored its potential application in enhancing plant tolerance to osmotic stress, and provided valuable insights for further elucidation of SlRCM1 function.