Mutagenesis is an effective strategy to generate genetic resources for functional genomics and crop improvement. It is widely accepted that chemical-induced mutations are randomly distributed across the genome. However, the randomness of such mutations remains a matter of scientific debate due to a lack of systematic investigation. Although the basic features of mutations induced by different mutagens are known, it remains unresolved whether these mutations correlate with the chromatin state and gene expression. In this study, genomes of the wild-type and 4,619 mutagenized M3 lines derived from treatment with two concentrations of ethyl methane sulfonate (EMS) are sequenced, identifying over 7 million single-nucleotide mutations (SNP^ms) that covered over 97% of genes in the rice genome. Comprehensive analyses integrated with genomic, epigenomic, and expression data revealed that SNP^ms are more frequently located in open chromatin regions and actively expressed genes. Within expressed genes, heterozygous SNP^ms are enriched both upstream and downstream of transcription start sites (TSSs), whereas homozygous SNP^ms are enriched significantly in the upstream regions. These findings demonstrate that EMS-induced mutations are non-randomly distributed across the rice genome, with a preference for functionally important genomic regions.