Animal mortality on roads is one of the main concerns on wildlife conservation. Due to their habitat requirements, amphibians became one of the most commonly road-killed group and this may affect their population viability. Implementation of mitigation measures may overcome the problem. However, due to the extensive road network, their application is very expensive and required a better understanding in where they should be implemented. Mortality hotspots can be identified as clusters of road-killed records) using GIS (Geographic Information Systems). Although there are several statistical methods available, it is lacking a comparison analysis of them in order to understand their pros and contras. The aim of this study was to analyse possible differences between global, multi-scale and local spatial analysis methods in defining hotspots using amphibian road fatality data collected in northern Portugal country roads. We calculated the Nearest neighbor index, Morans I and Getis-ord General in order to compare the global clustering of points in seven sampled roads, and three were identified as clustered. We used Ripley K-function, Ripley L-function and F function to calculate the best scale for Malo's equation and Kernel density analysis in detecting hotspots and we compared their detection performance with Local Indicators of Association (LISA) (i.e Local Moran's I and Getis-ord Gi). Three different GIS software applications were used: ArcGis, Quantum GIS with R (opensource) and GeoDa (opensource). Results showed the importance of using multidistance spatial cluster analysis to define the best scale for hotspot detection with Malo´s equation and Kernel density analysis. Here we also suggest the advantages of Local Indicators of Association (LISA) for detecting clusters with the contribution of each individual observation (Local Morans I and Getis-ord Gi).