Significant attention has been given to the role played by non-hematopoietic cells in the immune organs, including the lymph nodes, in hopes of understanding the development, maintenance, and regulation of the immune system. However, the molecular and cellular characterization of non-hematopoietic cells is still in its infancy. Here we show that non-hematopoietic cells in mouse lymph nodes can be fractionated into previously unidentified subpopulations according to the transgenic reporter expression of alpha-smooth muscle actin (αSMA). αSMA(+) non-hematopoietic cells were predominantly detected in gp38(+)CD31(-) and gp38(-)CD31(-) cells. Molecular expression profiles suggest similarities between αSMA(+)gp38(+)CD31(-) and αSMA(-)gp38(+)CD31(-) subpopulations and dissimilarities between αSMA(+)gp38(-)CD31(-) and αSMA(-)gp38(-)CD31(-) subpopulations. The results indicate that αSMA is a useful marker for further understanding the molecular and cellular characteristics of non-hematopoietic cells in the lymph nodes.