Emerging evidence suggests a positive association between relevant aspects of one's psychological identity and physical activity engagement, but the current understanding of this relationship is primarily based on scales designed to assess identity as a person who exercises, leaving out essential aspects of physical activities (eg, incidental and occupational physical activity) and sedentary behavior. The goal of this study is to evaluate the validity of a new physical activity and sedentary behavior (PA/SB) identity scale using 2 independent samples of US adults. In study 1, participants answered 21 candidate items for the PA/SB identity scale and completed the International Physical Activity Questionnaire-Short Form (IPAQ-SF). Study 2 participants completed the same PA/SB identity items twice over a 1-week interval and completed the IPAQ-SF at the end. We performed factor analyses to evaluate the structure of the PA/SB identity scale, evaluated convergent validity and test-retest reliability (in study 2) of the final scale scores, and examined their discriminant validity using tests for differences in dependent correlations. The final PA/SB identity measure was comprised of 3 scales: physical activity role identity (F1), physical activity belief (F2), and sedentary behavior role identity (F3). The scales had high test-retest reliability (Pearson correlation coefficient: F1, r=0.87; F2, r=0.75; F3, r=0.84; intraclass correlation coefficient [ICC]: F1: ICC=0.85; F2: ICC=0.75; F3: ICC=0.84). F1 and F2 were positively correlated with each other (study 1, r=0.76; study 2, r=0.69), while both were negatively correlated with F3 (Pearson correlation coefficient between F1 and F3: r=-0.58 for study 1 and r=-0.73 for study 2; F2 and F3: r=-0.46 for studies 1 and 2). Data from both studies also demonstrated adequate discriminant validity of the scale developed. Significantly larger correlations with time in vigorous and moderate activities and time walking and sitting assessed by IPAQ-SF with F1, compared with F2, were observed. Significantly larger correlations with time in vigorous and moderate activities with F1, compared with F3, were also observed. Similarly, a larger correlation with time in vigorous activities and a smaller correlation with time walking were observed with F2, compared with F3. This study provided initial empirical evidence from 2 independent studies on the reliability and validity of the PA/SB identity scales for adults.