Serum (n = 1,636) and egg yolk (n = 802) samples collected from hens on four commercial egg farms in Florida were tested for the presence of specific antibodies to Mycoplasma gallisepticum in a commercially available enzyme-linked immunosorbent assay. No significant differences were noted between serum and egg yolk samples with respect to distribution of positive, suspect, and negative test results or for the mean sample/positive control ratio values of positive, suspect, and negative test results. A linear relationship between the distribution of positive and negative results and the age of the birds was observed for results obtained with both serum and egg yolk samples. On the basis of the results of this study, egg yolk samples can be used in lieu of serum samples to screen flocks for antibodies to Mycoplasma gallisepticum.