The purpose of this study was to analyze the effects of toddler's temperament and toddler-teacher relationship on their peer interaction, focusing on gender differences. A total of 221 toddlers(122 boys and 99 girls) attending 16 child care centers in South Korea participated in this study. Each toddler was observed during a free-play period in the classroom over 4 sessions of 5 minutes, so a total of 20 minutes was measured. Two observers scored recorded observation scenes with two peer interaction criteria, including interaction initiation and interaction participation level. The main results of this study are as follows: First, there was a signigicant gender difference in peer interaction and toddler's temperament and toddler-teacher relationship. Second, the factor of toddler-teacher relationship significantly predicted the boys' peer interaction, while for the girls, the significant predictor was intentional control of temperamental factors. Further, implications for different approaches according to the gender difference of the affecting factor on toddlers' peer interaction have been discussed.