Objective: To understand comprehensive sexuality education knowledge among junior high school students in China, and to analyze relevant influencing factors to provide scientific basis for the implementation of school based sexuality education or comprehensive sexuality education in the future. Methods: By using convenient sampling method, a cross sectional survey was conducted among 4 545 students at grade 1 to grade 3 from junior middle schools in Beijing, Liaoning, Sichuan, Chongqing, Yunnan, Henan, using an online electronic questionnaire during September to October 2021. The questionnaire included general demographic information, subjective social status, sex education received at home and school, bullying, as well as knowledge, attitude, practice and needs towards comprehensive sexuality education. Results: The average score of the comprehensive sexuality education knowledge was (12.21±3.10) points, which was converted to (71.82±18.21) points based on the percentage based system. The scores varied by grade, sex and sexuality education setting, significantly higher in grade 3 students (12.69±2.99), girls (12.28±3.01) ( P < 0.01), and students who have received sex education at home (12.67±2.88) and students who received sex education at school (12.63±2.91), as well as those who had actively searched for sex information online had a higher score (12.71±2.94) ( t =35.10, 28.78, 10.55, P <0.01).Further analysis using multiple linear regression and Logistic regression, "whether you have received sex education at home" "whether you have received sex education at school" "whether you have searched the Internet actively" and "whether it is necessary to carry out comprehensive sex education" are still correlated with the score of comprehensive sex education knowledge; "self reported bullying" was still associated with "subjective social status" score, correct rate of question 2, and "shyness and inferiority toward the development of secondary sexual characteristics" ( P <0.01). Conclusion The current comprehensive sexuality education knowledge among junior high school students needs to be improved. The level of implementation of sexuality education in different settings varies, but the implementation of sexuality education in schools can help improve students comprehensive sexuality education knowledge level.