Objective-To examine the DNA methylation status of the ABCB1 gene in tumor cells of dogs with lymphoma. Animals-27 dogs with multicentric B-cell high-grade lymphoma (19 chemotherapy-sensitive dogs and 8 chemotherapy-resistant dogs). Procedures-The DNA methylation profile of the CpG island of the ABCB1 gene was analyzed by use of bisulphite sequencing and real-time methylation-specific PCR assay in lymphoma cells. Quantitative reverse transcriptase PCR assay of the ABCB1 gene was conducted to measure the amount of mRNA. Correlation between the amount of ABCB1 mRNA and the methylation rate was examined. Results-The CpG island of the ABCB1 gene was hypomethylated in most dogs in both the chemotherapy-sensitive and-resistant groups. No significant difference was detected in the methylation rate between the 2 groups, and no significant correlation was detected between the methylation rate and the mRNA expression level. Conclusions and Clinical Relevance-Expression of the ABCB1 gene was not suppressed by hypermethylation of its CpG island in most dogs with lymphoma regardless of their chemotherapy sensitivity status.