Marek's disease (MD) vaccines are cell-associated and require special handling and care during administration. Vaccine dose is evaluated by plaque assay and is indicated as the number of plaque-forming units (PFUs) per dose. The objectives of this study were to evaluate the dose variability within each vial of MD vaccines and to assess those factors (from both manufacturing and handling and administration of the vaccine) that could affect vaccine dose variability. Three experiments were conducted. Experiment 1 was to evaluate dose variability in 36 MD vaccine vials and the effect of manufacturing factors on dose variability. Vaccines were titrated 10 times. Dose variability was measured as the coefficient of variability (CV) calculated as standard deviation divided by average PFU and multiplied by 100. Our results showed that all evaluated vaccines had levels of CV ranging from 10% to 34%. Variability existed regardless of manufacturer, vaccine serotype, and batch. Experiment 2 was conducted to evaluate the effect of infectivity rate (IR) on CV. IR was artificially reduced by adding noninfected chicken embryo fibroblast to the reconstituted vaccine before titration. Our results showed that decreased IR results in higher CV. Experiment 3 was to evaluate the handling and administration factors (time and mixing during administration) on CV. Our results showed that CV tends to increase with time and that this effect is more remarkable if vaccines were not mixed. Our study emphasizes the relevance of proper handling of MD vaccines and shows that dose variability can jeopardize the uniformity of vaccination in a flock and therefore the success of vaccination.