Although cycling has been associated with overuse/fatigue and acute injuries, there is lack of information regarding associated risk factors and prevention factors. The objective of the study was to determine the factors associated with injury, and perceptions of discomfort and pain in cyclists. A total of 739 cyclists completed an online questionnaire between February and October 2016. The questionnaire acquired information on participant demographics, characteristics related to cycling profile and fitness training, bike components and cycling posture, self-reported perceptions of comfort and pain, and injuries sustained in the last 12 months. Logistic regression models estimated odds ratios (OR) and 95% confidence intervals (95%CI) that examined factors associated with reporting overuse/fatigue injury, acute injury, body discomfort, saddle discomfort, and pain while cycling. Odds of reporting an overuse/fatigue injury increased when the cyclists complemented training with running (OR = 1.74; 95%CI = 1.03-2.91) or swimming (OR = 2.17; 95%CI = 1.19-3.88), and with reported pain while cycling (OR = 1.17; 95%CI = 1.05-3.69) and not cycling (OR = 1.76; 95%CI = 1.07-2.90). Odds of reporting an acute injury increased when biking to work (OR = 1.79; 95%CI = 1.07-2.86), and decreased with increased average cycling speed (1-km/h decrease OR = 0.93; 95%CI = 0.88-0.97), and compared to low-end bike, with the use of mid-range (OR = 0.25; 95%CI = 0.09-0.72) and high-end bike (OR = 0.34; 95%CI = 0.13-0.96). Although body discomfort was only associated with saddle discomfort and the presence of pain during cycling, saddle discomfort was also associated with biking to work (OR = 0.46; 95%CI = 0.22-0.88). Finally, pain perception was associated with a number of factors such as ride to work, core training, cycling experience, saddle discomfort, pain while not cycling. Numerous factors are associated with injury, and perceptions of discomfort and pain in cyclists. Such factors should be considered when developing training routines, bicycle maintenance best practices, and injury prevention programs.