Background: Cycle skills training (CST) improves children’s cycling knowledge and skills. This study examined and compared the effects of short-term CST (1-10 weeks) with or without on-road training on cycling-related knowledge, confidence and behaviours in adolescent girls. Methods: Girls (n = 117; age: 13.9 +/- 0.7 years) participated in either playground-based CST only (Traffic-Free CST; n = 43) or combined playground-based and on-road CST (Traffic-Free +OnRoad CST; n = 74). Participants completed pre-training and post-training surveys about cycling-related knowledge, confidence and behaviour, and practical cycling skills assessment (fundamental and advanced skills). Data were analysed using paired t-test and McNemar tests. Results: At baseline, few adolescents cycled > 1/week (11.1%) or to school (2.6%). Both types of CST improved adolescents’ knowledge (Traffic-Free: 82.9 +/- 13.0% to 88.9 +/- 9.5%, p = 0.001; Traffic-Free + OnRoad: 85.9 +/- 8.8% to 93.6 +/- 6.4%, p < 0.001). Traffic-free CST improved adolescents' confidence to cycle in the parks/playgrounds (very confident: 41.9% to 60.5%; p = 0.034). Traffic-Free+ OnRoad CST increased adolescents' confidence to cycle on the road (very confident: 41.9% to 54.1%; p = 0.013), but not to school (very confident: 25.4% to 31.5%; p = 0.146). Cycling habits and preferences did not change significantly after CST (all p > 0.05). Traffic-Free+ OnRoad CST participants were competent in most fundamental (97.8 +/- 7.6%) and advanced practical cycling skills (97.6 +/- 5.7%), whereas Traffic-Free CST participants had lower scores in fundamental skills (81.4 +/- 29.2%; p < 0.001). Conclusion: CST with or without on-road training improved cycling-related knowledge but did not change cycling habits in adolescent girls. CST with on-road training improved adolescent girls' confidence to cycle on the road, but not to school. Future CST programs should be tailored to the adolescents' needs and preferences.