Vehicle-tree collisions are the most common type of road crash with fixed obstacle in Czech Republic. Based on the literature review and using real world in-depth crash data, this paper aims to define factors, which significantly influence the injury severity of single vehicle-tree crashes. In-depth data provides a comprehensive view to the failure on the system infrastructure-human-vehicle related to crash. The in-depth crash database includes very detailed information related to infrastructure, vehicle, human failure and crash participants characteristics and their medical condition and also crash reconstruction. Multinomial logistic regression and generalized linear mixed model were used to determine the individual effect of each predictor. The statistically significant variables were the day period, trunk diameter and impact speed. Using multinomial logistic regression shows also vehicle age as statistically significant. Obtained results can help to efficiently direct countermeasures not only on the road infrastructure—e.g. speed reduction in selected locations with specified tree character. However, the emphasis should be also focused on driver behaviour.