The yak (Bos grunniens) exhibits exceptional regional adaptability, enabling it to thrive in the distinctive ecological niches of the Qinghai–Tibet Plateau. Its survival relies on the intricate balance of its intestinal microbiome, essential for adapting to harsh environmental conditions. Despite the documented significance of bacteria and fungi in maintaining intestinal homeostasis and supporting immune functions, there is still a substantial gap in understanding how the composition and functionality of yak gut microbiota vary along altitude–temperature gradients. This study aims to fill this gap by employing 16S rRNA and ITS amplicon sequencing techniques to analyze and compare the intestinal microbiome of yaks residing at different elevations and exposed to varying temperatures. The findings demonstrate subtle variations in the diversity of intestinal bacteria and fungi, accompanied by significant changes in taxonomic composition across various altitudes and temperature gradients. Notably, Firmicutes, Actinobacteriota, and Bacteroidota emerged as the dominant phyla across all groups, with Actinobacteriota exhibiting the highest proportion (35.77%) in the LZF group. Functional prediction analysis revealed significant associations between the LZF group and metabolic pathways related to amino acid metabolism and biosynthesis. This suggests a potential role for actinomycetes in enhancing nutrient absorption and metabolism in yaks. Furthermore, our findings suggest that the microbiota of yaks may enhance energy metabolism and catabolism by modulating the Firmicutes-to-Bacteroidota ratio, potentially mitigating the effects of temperature variations. Variations in gut bacterial and fungal communities among three distinct groups were analyzed using metagenomic techniques. Our findings indicate that microbial genera exhibiting significant increases in yaks at lower altitudes are largely beneficial. To sum up, our research investigated the changes in gut bacterial and fungal populations of yaks residing across diverse altitude and temperature ranges. Moreover, these results enhance comprehension of gut microbial makeup and variability, offering perspectives on the environmental resilience of dry lot feeding yaks from a microbial angle.