Chlorophyll content and fluorescence parameters are crucial indicators to evaluate the light use efficiency in rice; however, the correlations among these parameters and the underlying genetic mechanisms remain poorly understood. Here, to clarify these issues, we conducted a genome-wide association study (GWAS) on 225 rice accessions. In the phenotypic and Mendelian randomization (MR) analysis, a weak negative correlation was observed between the chlorophyll content and actual quantum yield of photosystem II (ΦII). The phenotypic diversity observed in SPAD, NPQt, ΦNPQ, and Fv/Fm among accessions was affected by genetic background. Furthermore, the GWAS identified 78 SNPs and 17 candidate genes significantly associated with SPAD, NPQt, ΦII, ΦNPQ, qL and qP. Combining GWAS on 225 rice accessions with transcriptome analysis of two varieties exhibiting distinct fluorescence characteristics revealed two potential candidate genes (Os03g0583000 from ΦII & qP traits and Os06g0587200 from NPQt trait), which are respectively associated with peroxisomes, and protein kinase catalytic domains might involve in regulating the chlorophyll content and chlorophyll fluorescence. This study provides novel insights into the correlation among chlorophyll content and fluorescence parameters and the genetic mechanisms in rice, and offers valuable information for the breeding of rice with enhanced photosynthetic efficiency.