Effects of F-strain Mycoplasma gallisepticum (FMG) inoculation and 1.5% supplemental dietary poultry fat (PF) on the egg yolk characteristics of commercial layers at 24, 34, 44, 50, and 58 wk of age were investigated. Sham and FMG inoculations were administered at 12 and 22 wk of age and dietary treatments (basal control and basal control with 1.5% supplemental PF) were initiated at 20 wk of age. Yolk lipid concentration was reduced on wk 24 in birds that had been inoculated at 12 or 22 wk of age with FMG. The use of 1.5% supplemental PF increased percentage of yolk weight and yolk:albumen ratio across age and inoculation treatment. At 58 wk of age, concentrations of yolk palmitic acid increased and those of oleic and linolenic acid decreased when sham inoculations were given at 22 rather than at 12 wk of age. However, FMG inoculations given at 22 rather than at 12 wk increased palmitoleic acid and decreased stearic acid yolk concentrations. At 12 wk of age, FMG inoculations decreased yolk palmitoleic, oleic, and linolenic acid concentrations while causing increased yolk stearic and arachidonic acid levels when compared with sham inoculations. Furthermore, 1.5% supplemental PF decreased concentrations of palmitic and oleic acid and increased those of linoleic acid in the yolk at 58 wk of age. Despite the interaction of 1.5% supplemental PF with the prelay inoculation of FMG on early (18 to 26 wk) layer performance noted in a previous report, the effects of a prelay FMG inoculation and 1.5% supplemental PF on the egg yolk characteristics examined in the current study were independent of each other. This suggests that 1.5% supplemental PF is not effective in modulating the effects of an FMG inoculation at 12 wk of age on hen egg yolk characteristics between 24 and 58 wk of age and that the combined effects of PF supplementation and FMG inoculation on performance do not influence egg yolk characteristics.