There are no data currently available on gender and racial variation in smallpox vaccine immune responses. We recruited 1076 healthy adults 18-40 years old who received one dose of the US-licensed smallpox vaccine (Dryvax). Vaccinia neutralizing antibody titers in each subject's serum were determined using a high throughput neutralization assay based on a recombinant, beta-gal expressing vaccinia virus. Results are reported as the serum dilution inhibiting 50% of virus activity (ID(50)). The median ID(50) for all subjects was 132.2 (inter-quartile range (IQR)=78.8, 205.6). While no significant differences were observed with race and ethnicity, females had significantly higher neutralizing antibody titers than males (158.5 [93.2, 255.8] vs. 124.1 [75.2, 185.9]; p<0.0001). As expected, time since vaccination was also associated with variations in neutralizing antibody titers in our subjects. These data indicate that neutralizing antibody titers following primary smallpox vaccination vary by gender.