Peripheral blood mononuclear cells (PBMCs) offer a significant promise for gene expression analyses as a substitute for tissues that are not easily accessible. The objective of this study was to validate the use of PBMCs for gene expression analysis as a marker of nutritional intervention as an alternative to skeletal muscle tissue (SMT) biopsies. We performed a transcriptome comparison of PBMCs versus SMT after an 8-week supplementation with n-3 polyunsaturated fatty acid (PUFA) in 16 obese and insulin-resistant subjects. Expression levels of 48,803 transcripts were assessed by the Human-6 v3 Expression BeadChips (Illumina, San Diego, CA). In SMT, 36,738 (75%) transcripts were detected, whereas 34,182 (70%) transcripts were detected in PBMCs. Further, 88% (32,341) of these transcripts were coexpressed in both tissues. Importantly, a strong correlation (r = 0.84, p < 0.0001) was observed between transcript expression levels of PBMCs and SMT after n-3 PUFA supplementation. In conclusion, PBMCs express the majority of transcripts expressed in SMT subsequent to n-3 PUFA supplementation and their expression levels are comparable. In the interest of practicalities and cost, these results support the use of PBMCs as a surrogate model for SMT gene expression in nutrigenomic studies. Further research on PBMC and SMT gene expression in response to other nutritional exposures is warranted.