Previous studies from the US have found that female physicians often experience gender-based discrimination related to professional advancement. In Japan, female physicians are underrepresented in leadership positions but little is known about the prevalence of gender discrimination. We investigated the perception and prevalence of gender-based career obstacles and discrimination among Japanese physicians. The study was based on surveys of alumnae from 13 medical schools and alumni from 3 medical schools. In total, 1,684 female and 808 male physicians completed a self-administered questionnaire (response rate 83% and 58%). More women than men had the perception of gender-based career obstacles for women (77% vs. 55%; p < 0.0001). Women with part-time positions were more likely to have the perception of gender-based career obstacles than women working full-time (OR 1.32, 95% CI: 1.01-1.73). More women than men reported experience of gender discrimination related to professional advancement (21% vs. 3%; p < 0.0001). Factors associated with experience of gender discrimination included age (p < 0.0001), marital status (p < 0.0001), academic positions (p < 0.0001), subspecialty board certification (p = 0.0011), and PhD status (p < 0.0001). Women older than 40 years were more likely to experience gender discrimination compared with younger women (OR 5.77, 95% CI: 1.83-18.24 for women above 50, and OR 3.2, 95% CI: 1.48-7.28 for women between 40 and 49) and women with PhD were more likely to experience gender discrimination (OR 4.23, 95% CI: 1.81-9.89). Our study demonstrated that a significant proportion of Japanese women experienced gender-based discrimination and perceived gender-based career obstacles compared with male physicians.