Habitat fragmentation due to transport infrastructure and other human development poses a threat to many wildlife species. This threat may differ depending on the species and types of fragmenting elements. There is a need to quantify the level of habitat fragmentation and the impact of habitat fragmentation on different wildlife species for use in transportation planning. Such measures would be useful in assessing the cumulative impacts of multiple road projects on wildlife connectivity and habitat suitability, for long-range wildlife impact mitigation planning for transportation projects, and as an indicator for the environmental monitoring of habitat fragmentation due to roads. Effective mesh size (meff) is a biologically relevant landscape metric that quantifies the degree of landscape fragmentation. The definition of the effective mesh size is based on the probability that two randomly chosen points in a region will be located in the same non-fragmented area of land. We calculated effective mesh size to assess the level of land¬scape fragmentation in the State of California, USA, based on four fragmentation geometries defined by a combination of highways, minor roads, urbanized areas, agricultural areas, and natural fragmenting features (e.g., rivers, lakes, and alpine areas). The effective mesh size for these four fragmenting geometries were calculated for the entire State of California using eight sets of planning units: 1) transportation planning districts, 2) municipal county boundaries, and 3) six levels of watersheds. To demonstrate the methodology, we examined how effective mesh size may impact two species important to transportation planning in California: mule deer (Odocoileus hemionus) and mountain lion (Puma concolor). The calculated effective mesh sizes were compared with the home range sizes and daily movement distances of the selected focal species to determine the potential impacts of habitat fragmentation and to identify areas where transportation projects will potentially impact these focal species. Based on the results of this analysis, we suggest that integrating an effective mesh size-based tool into transportation planning frameworks would be valuable to improve identification of potential landscape level impacts early in the planning process. The calculation of effective mesh size will give transportation planners a way to analyze the cumulative impacts of roads in districts, counties, and watersheds and can be used as an environmental indicator for ecological assessment of transportation system impacts.