Epigenetic modifications of plants, such as DNA methylation, are often regulated by environmental stress. P450 monooxygenases play an important role in plant growth and development, and often respond to environmental stimuli. However, little is known about the mechanism that regulates P450 gene expression changes upon abiotic stress. Here, we tested the hypothesis that P450�s responds to temperature stress through changes in gene body methylation. A P450 gene that was up-regulated under heat and cold treatments was cloned from perennial ryegrass, and named LpCYP72A161 according to the amino acid sequence homology. LpCYP72A161, which contained four exons and three introns, showed CG methylation, and the methylated sites were mainly located on CpG islands within exon 1 and 4. Temperature stress resulted in a significant reduction in methylation of CG sites on exon 1 but not other CG islands. We observed that overlapping mCGs sites were demethylated upon heat and cold treatment; however, heat-treatment caused a higher demethylation and higher expression of LpCYP72A161 compared with cold treatment. In addition, plants that were recovered from heat treatment showed partial recovery of methylation and expression. In conclusion, LpCYP72A161, a CG-methylated gene, exhibited a flexible shift of gene body methylation in exon 1 under heat and cold stress, and the methylation level was negatively correlated to transcript abundance. By analyzing the changes in methylation patterns of the P450 gene LpCYP72A161 during early response to temperature stress in perennial ryegrass, our research provides a new understanding of the regulation of the gene body methylation in temperature stress.