Abstract Gut microbiota variations in response to environmental and nutritional factors are of great significance as gut microbiota plays an integral role in nutrient metabolism, immunity, health, and disease conditions. In this context, limited studies investigated variations of gut microbiota in response to different feeding systems and environmental conditions. The current study obtained fresh fecal samples from house-fed (LS) and grazing yaks (LF) from Linzhou County. 16 S rRNA amplicon sequencing of the V3–V4 and internal transcribed spacer 2 (ITS2) domains generated 16,332 bacterial and 2345 fungus amplicon sequence variants (ASVs). Alpha and beta diversity indices revealed significant variations (p > 0.05) in gut microflora between the two groups. At the phylum level, Firmicutes, Actinobacteriota, Bacteroidota, and Patescibacteria regarding bacteria, and Ascomycota and Basidiomycota regarding fungi dominated. At the genus level, UCG-005, Rikenellaceae_RC9_gut_group, Clostridium_sensu_stricto_1, g__Muribaculaceae, UCG-010, [Eubacterium]_coprostanoligenes_group, Turicibacter, Alistipes, Prevotellaceae_UCG-003, UCG-009, Blautia, dgA-11_gut_group, Candidatus_Saccharimonas dominated in LS, while Anthrobacter and Terrisporobacter dominated in the LF group. Fungal genera like Myrothecium and Plectosphaerella dominated the LS group, while Neoascochyta, Paraphaeosphaeria, and Hypocreales dominated the LF group. Also, significant variations (p > 0.05) in gene expressions were found between the two groups. These findings provide insights into yak gut microbiota adaptations and metabolic changes in response to varied environmental conditions and can provide valuable information, optimizing feeding strategies after identifying specific differences between grazing and house-fed yaks, reducing environmental impacts, and improving yaks’ health and productivity in specific geographical settings.