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Background: Road traffic injuries (RTI) are a major public health epidemic killing thousands of people daily. Low and middle-income countries, such as Brazil, have the highest annual rates of road traffic fatalities. In order to improve road safety, this study mapped road traffic fatalities on a Brazilian highway to determine the main environmental factors affecting road traffic fatalities., Methods and Findings: Four techniques were utilized to identify and analyze RTI hotspots. We used spatial analysis by points by applying kernel density estimator, and wavelet analysis to identify the main hot regions. Additionally, built environment analysis, and principal component analysis were conducted to verify patterns contributing to crash occurrence in the hotspots. Between 2007 and 2009, 379 crashes were notified, with 466 fatalities on BR277. Higher incidence of crashes occurred on sections of highway with double lanes (ratio 2∶1). The hotspot analysis demonstrated that both the eastern and western regions had higher incidences of crashes when compared to the central region. Through the built environment analysis, we have identified five different patterns, demonstrating that specific environmental characteristics are associated with different types of fatal crashes. Patterns 2 and 4 are constituted mainly by predominantly urban characteristics and have frequent fatal pedestrian crashes. Patterns 1, 3 and 5 display mainly rural characteristics and have higher prevalence of vehicular collisions. In the built environment analysis, the variables length of road in urban area, limited lighting, double lanes roadways, and less auxiliary lanes were associated with a higher incidence of fatal crashes., Conclusions: By combining different techniques of analyses, we have identified numerous hotspots and environmental characteristics, which governmental or regulatory agencies could make use to plan strategies to reduce RTI and support life-saving policies.