Protein kinase plays important roles in plant growth, development, and responses to various biotic and abiotic stresses. Levels of nucleotide diversity, however, have not been investigated to date in natural populations of wild rice. In this study, the diversity of the MAPK5 gene and its characteristics were analyzed in samples of wild rice collected from natural populations in Thailand and Lao PDR, using a re-sequencing DNA technique. For the entire of 514 nucleotides sequence analyzed, non-synonymous, synonymous substitutions, and frame-shift indels were identified in coding sequence of exon1, but not for exon2. For non-coding sequences, indel mutations were present in the sequenced region of the first intron of the MAPK5 gene. These results suggest that alternative splicing transcripts of the MAPK5 gene are potentially generated in response to various environment factors. There was substantial nucleotide variation with a high estimate of nucleotide diversity (pi = 0.18113), number of haplotypes (H = 15), and the number of polymorphic sites (S = 319). The Tajima's D value of the MAPK5 gene was 1.673 (p > 0.10), suggesting that the MAPK5 gene of wild rice evolved neutrally. The high level of nucleotide diversity found in natural populations of wild rice may provide populations with the opportunity to adapt to environmental changes in their natural habitats.