The frequency of Mycoplasma synoviae exposure in a convenience sample of commercial layers was established by the presence of antibody in eggs. Chloroform-extracted egg yolks were found to be more suitable than saline-extracted yolks, and were used with a commercial enzyme-linked immunosorbent assay kit, having first established the sensitivity and specificity of the kit with eggs from known M. synoviae-positive and M. synoviae-negative flocks. For the prevalence study, pooled yolks from 12 eggs were obtained from each of 56 randomly selected laying farms in possession of a packing station number in the east of England. This number allowed 95% confidence of detection of antibody in one egg if present in 50% of the flock. Eggs were taken from the oldest flock on each site and were returned with a completed questionnaire. The prevalence of egg antibody to M. synoviae was 78.6% (95% confidence interval, 65.6, 88.4). This study has confirmed that chloroform extraction of yolk antibody is a suitable approach for assessing the flock prevalence of M. synoviae infection in layer hens.