Objectives The Sasang Personality Questionnaire (SPQ) measures the Yin-Yang temperament of Sasang typology with proven structural and clinical validity in adults, however it was not examined with teenagers. The purpose of this study was to validate the biopsychological structure of SPQ in middle school students. Methods 681 Korean high school students (356 boys and 325 girls) completed SPQ, Junior version of Temperament and Character Inventory (JTCI), and height and weight measures. The correlation between SPQ and JTCI subscales were examined, and the differences of SPQ and JTCI subscales, Body Mass Index (BMI) and Pondera Index (PI) among high (30%), middle (40%), and low (30%) SPQ total score groups were investigated with Analysis of Variance (ANOVA). The profile analysis was also performed to compare JTCI subscale profiles of three SPQ total score groups. Results The SPQ score was significantly (p<0.001) correlated positively with JTCI Novelty-Seeking and negatively with JTCI Harm-Avoidance. The JTCI Novelty-Seeking score of high SPQ group was significantly (p<0.001) higher than that of low SPQ group, and the JTCI Harm-Avoidance score of low SPQ group was significantly higher than that of high SPQ group. The JTCI subscale profiles for three SPQ groups were significantly different for boys and girls. Significant correlations or differences for the PI and BMI among SPQ groups were not found. Conclusions This study presented that the biopsychological structure of SPQ is robust as shown in adults. The SPQ would be a useful clinical measures of Sasang typology in pediatric patients.