Background Previous human papillomavirus (HPV) and HPV vaccine knowledge scales have focused on young adults in the vaccination catch-up age range or parents of vaccine eligible adolescents. Previous scales are not specific to the new guidelines for HPV vaccination in mid-adults. The study aimed to develop and validate a mid-adult HPV vaccine knowledge scale informed by the latest vaccine recommendations. Methods Self-reported data were collected using a cross-sectional survey of adults aged 27 to 45 years with no history of HPV vaccination (n = 706). Exploratory and confirmatory factor analyses identified latent constructs in a 13-item mid-adult HPV vaccine knowledge scale. Sociodemographic differences in the HPV vaccine knowledge were also assessed. Results The mean of correct responses across all items was 5.9 (SD, 2.8; range, 0-13). Exploratory and confirmatory factor analyses revealed a 3-factor structure best explained the data with a good construct validity and reliability. The first factor contained 6 items about HPV infection, the second factor contained 3 items about HPV prevention through vaccination, and the third factor contained items about HPV vaccination misinformation. Analysis of variance and t test found significant group level differences in knowledge among sex, race, educational level, annual income, health insurance, and marital status. Conclusions Our study supports the use of a knowledge scale for US mid-adults to assess HPV and HPV vaccination knowledge because the patient requires some baseline knowledge of this recommendation to discuss the vaccine with their health care provider. The mid-adult HPV vaccine knowledge scale can measure basic HPV knowledge important to informed decision making.