Naturally occurring antibodies (NAbs) are reliable biomarkers of baseline humoral immunity in poultry raised under diverse production housing systems. To validate and extend prior observations, we compared quantitative and qualita-tive profiles in IgM and IgY NAbs between free-range and conventional broiler chicks sampled at matched ages. Sera were collected from three farms (n = 144): conventional broiler (Ross 308) at 47 days (C-47), and free-range (Sasso) at 47 (FR-47) and 67 days (FR-67). NAb measurements were performed by indirect ELISA against actin, DNA, TNP–BSA, and LPS before and after dissociation assays (urea and heat-induced cryptic activity), alongside serum total IgM/Y class quantification. We found consistently higher IgM NAb levels in free-range broilers, and normalization to total IgM further confirmed these differences, supporting qualitative binding features between the two genotypes (p < 0.001). Persistent IgY patterns were antigen-specific: higher anti-LPS (~1.2-fold) and lower anti-DNA (~0.6-fold) in free-range broilers. While total IgM was higher in free-range groups, total IgY did not differ in concentration, although dissociation assays showed multiple qualitative differences. These findings further validate our previous observations and demon-strate that both, antibody levels and binding characteristics distinguish broiler genotypes, supporting the utility of NAbs as practical biomarkers for monitoring immune status and informing management decisions in commercial poultry production.