The fragmentation of habitats and the creation of barriers caused by traortation infrastructures reduce landscape connectivity, which is suspected to be one of the most important factors causing population declines. Road networks affect wildlife habitats in two different ways. First, road construction leads to a direct habitat loss. Second, maintenance and use of road cause various effects altering habitat quality, such as increasing access to hunters, poachers and tourists. Development of roads through protected areas and ecological sensitive regions can have catastrophic effects on wildlife and leaves destructive impacts such as habitat fragmentation and isolation. Notwithstanding these negative ecological impacts, road networks continue to increase worldwide due to increasing socio-economic needs for high mobility. In Iran, road construction in sensitive habitats, protected areas and national parks is expanding during the past decades. This study focuses on the ecological impacts of Isfahan’s west freeway, which passes through Ghamishloo wildlife refuge, I.U.C.N category IV, in Isfahan Province. The two key affected species of the study area goitered gazelle (Gazella subgutturosa subgutterosa) and wild sheep (Ovis orientalis isfahanica) were considered for impact analysis. We used HEP (Habitat Evaluation Procedure) as a habitat-based impact assessment methodology which considers habitat quality and quantity. Habitat quality was measured as habitat suitability index for each species. By exhaustive literature review and field observations, six effective variables on habitat suitability including vegetation cover, slope, elevation, distance to water and distance to road were identified and the suitability maps was generated for each variable by assigning a suitability index (scale 0-1) to conditions for each variable. By including the equation for calculating the habitat suitability index (HSI), the final habitat suitability map for the study species were generated. Habitat unit (HU) was derived from multiplying the HSI for each of evaluation species by the habitat area (HU = HSI × size of habitat) in two time (before and after road construction). The results showed that due to the presence of the freeway, 7710 (HUs) for Gazella subgutturosa subgutterosa and 6288 (HUs) for Ovis orientalis isfahanica have lost. In addition, for quantifying landscape pattern change due to road construction, various landscape metrics such as PLAND (Percentage of Landscape), NP (Number of Patches), ED(Edge Density), MPS (Mean Patch Size), LPI (Largest Patch Index), MNN (Mean Nearest Neighbor), and CONTAG (Contagion) were calculated for the species distribution polygons for two time before and after road construction. Analysis of landscape metrics revealed increasing distance between available habitat patches, the additive barrier of roads, limit dispersal possibilities of species and decrease landscape connectivity in the study area, in particular due to absence of wildlife crossing structures such as underpasses and over passes. Our result provided quantitative data on habitat loss and landscape fragmentation in Ghamishloo wildlife refuge and indicated negative impacts of the freeway on wild sheep and Goitered gazelle populations by restricting movement between habitat patches in Ghamishloo wildlife refuge, and presents a concern for the conservation of these vulnerable species. According to the results of this study, mitigation and compensation activities should be considered in Ghamishloo wildlife refuge. Keywords: Ecological Impact assessment, Traortation Infrastructure, Habitat fragmentation, Gazella subgutturosa, Ovis orientalis isfahanica, HEP, Habitat Suitability Index, LandscapeMetrics