Global warming and climate change have been recognized as key factors contributing to the decline in insect pollinator populations. Understanding how animals can quickly respond to temperature changes is essential for predicting the impacts of climate change on biodiversity. An adequate microclimate is crucial for determining habitat quality for butterflies, particularly for the less mobile immature stages. Butterflies, like other insects, rely on behavioral thermoregulation, with some species being more vulnerable to the effects of climate change. In this study, conducted at 33 selected localities in the Republic of Serbia, an assessment was carried out to determine the impact of key environmental variables on the abundance and species composition of butterflies. In total, 105 butterfly species (Lepidoptera: Rhopalocera) were registered and analyzed during three seasons (spring, summer, and autumn) in 2022. To evaluate the effect of selected environmental and landscape variables on the abundance and species composition of butterflies, we used generalized linear models and canonical correspondence analyses (CCAs). To identify the most significant environmental variables influencing species composition and the abundance of butterflies, a “forward selection” analysis was conducted which showed a high dependence of the examined species on green cover and temperature. Furthermore, Van Dobben circles (t value biplots) were used to assess the significance of specific species' preference for temperature. The results showed that butterfly species which demonstrated preferences for lower values of temperature compared to other species were as follows: Erebia medusa, Melitaea arduinna, Melitaea trivia, Brenthis ino, Callophrys rubi, and Pseudophilotes vicrama. Two of these species are listed as strictly protected in the Republic of Serbia, and one is in the IUCN Nearly Threatened category in Europe. Given the results, species with preferences for lower temperatures may experience population decline in the future.