Genome-resolved metagenomics, based on shotgun sequencing, has become a powerful strategy for investigating animal-associated bacterial communities, due its heightened capability for delivering detailed taxonomic, phylogenetic, and functional insights compared to amplicon sequencing-based approaches. While genome-resolved metagenomics holds promise across various non-lethal sample types, their effectiveness in yielding high-quality metagenome-assembled genomes remains largely unexplored. Our investigation of fecal and cloacal microbiota of the mesquite lizards (Sceloporus grammicus) using genome-resolved metagenomics revealed that fecal samples contributed 97% of the 127 reconstructed bacterial genomes, whereas only 3% were recovered from cloacal swabs, which were largely enriched with host DNA. Taxonomic, phylogenetic and functional alpha bacterial diversity was greater in fecal samples than in cloacal swabs. We also observed significant differences in bacterial community composition between sampling methods, and higher inter-individual variation in cloacal swabs. Bacteroides, Phocaeicola and Parabacteroides (all Bacteroidota) were more abundant in the feces, whereas Hafnia and Salmonella (both Pseudomonadota) increased in the cloaca. Functional analyses showed that metabolic capacities of the microbiota to degrade polysaccharides, sugars and nitrogen compounds were enriched in fecal samples, likely reflecting the role of intestinal bacteria in nutrient metabolism. Overall, our results indicate that fecal samples outperform cloacal swabs in characterizing bacterial assemblages within lizards using genome-resolved metagenomics.