Background: Road traffic injuries (RTIs) are a substantial cause of mortality and disability globally. There is little published information regarding healthcare resource utilization following RTIs, especially in low and middle-income countries (LMICs). The aim of this study was to assess total hospital charges and length of stay (LOS) associated with RTIs in Iran and to explore the association with patients' socio-demographic characteristics, insurance status and injury-related factors (e. g. type of road users and safety equipment). Method: The study was based on the Iranian National Trauma Registry Database (INTRD), which includes data from 14 general hospitals in eight major cities in Iran, for the years 2000 to 2004. 8,356 patients with RTI admitted to the hospitals were included in the current study. The variables extracted for the analysis included total hospital charges and length of stay, age, gender, socio-economic and insurance status, injury characteristics, medical outcome and use of safety equipment among the patients. Univariable analysis using non-parametric methods and multivariable regression analysis were performed to identify the factors associated with total hospital charges and LOS. Results: The mean hospital charges for the patients were 1,115,819 IRR (SD=1,831,647 IRR, US$128 +/- US$210). The mean LOS for the patients was 6.8 (SD =8 days). Older age, being a bicycle rider, higher injury severity and longer LOS were associated with higher hospital charges. Longer LOS was associated with being male, having lower education level, having a medical insurance, being pedestrian or motorcyclist, being a blue-collar worker and having more severe injuries. The reported use of safety equipment was very low and did not have significant effect on the hospital charges and LOS. Conclusion: The study demonstrated that the hospital charges and LOS associated with RTI varied by age, gender, socio-economic status, insurance status, injury characteristics and health outcomes of the patients. The results of the study provide information that can be of importance in the planning and design of road traffic injury control strategies.