To determine the role of genetics in baseline lymphocyte parameters, several distinct lines of chickens were examined for differences in peripheral blood leukocyte (PBL) populations. Four highly inbred chicken lines (MHC congenic Fayoumi lines M15.2 and M5.1, and MHC congenic Leghorn lines G-B1 and G-B2), two advanced intercrosses [F5 (Broiler × G-B2) and F5 (Broiler × M15.2)], and an outbred population of broilers were used. Leukocytes isolated from healthy adult birds were labeled with monoclonal antibodies: chCD3, chCD4, chCD8, chBu-1, and hCD14. Flow cytometry was used to determine the total percentage of positively labeled cells for each surface marker in a sample, as well as the mean fluorescent intensity, or surface marker density, of a labeled subset. Significant line differences for percentage positive CD3 T cells and the ratio of B cells:T cells (represented by the Bu-1:CD3 ratio) were found. The effect of line was also significant for CD3 and CD8 T cell receptor density. Effects of sex and MHC on PBL cell surface marker expression were not significant in the lines examined. This study demonstrates the effect of genetic line on resting leukocyte composition of peripheral blood in the chicken lines examined. Observed PBL differences add to our growing knowledge of the varied roles that immune system status (defined by specific cell populations) and genetic background have in determining susceptibility and disease progression in chickens.