Recently, we have reported trained innate immunity in laying chicken monocytes. In the present study, we further investigated trained innate immunity of monocytes in layers and broilers. Monocytes of both breeds isolated from blood were trained in vitro with β-glucan, rec-chicken IL-4 or a combination of both, and restimulated with lipopolysaccharide (LPS), after which inflammation and metabolism-related responses were measured. Training of laying and broiler hen monocytes resulted in increased mRNA levels of IL-1β, iNOS and HIF-1α, but enhanced surface expression of CD40 and NO production was only observed in layers. Our in vitro study demonstrates that monocytes from different genetic backgrounds can be trained. However, the observed differences suggest a differential effect on immune functionality associated with innate training. Whether these differences in immune functions between layers and broilers have effect on disease resistance remains to be elucidated.