Microbial communities play crucial roles in human and environmental health. However, their emergent properties often arise not from individual bacteria but from spatially structured sub-assemblages that drive ecosystem dynamics and microbial interactions. These spatial niches influence key processes such as pathogen colonization, antibiotic resistance, and nutri-ent cycling, making their study essential for addressing infectious diseases, antimicrobial re-sistance, and climate-driven ecosystem shifts. Despite their importance, most spatial analysis approaches fail to resolve fine-scale microbial organization. Conventional bulk metagenomics and 16S sequencing lose spatial context, while imaging-based methods, though powerful, re-main low-throughput and require extensive probe design. To overcome these limitations, we de-veloped Split-And-pool Metagenomics PLot-sampling sequencing (SAMPL-seq), a high-throughput spatial metagenomics technique that captures microbial co-localization at the micron-scale. In this talk, I present the comprehensive SAMPL-seq method for the human and animal gut microbiome, as well as its application to complex environmental samples such as soil, bio-film, and mold-contaminated ecosystems. This method details sample preparation into micron-scale particles that retain spatial structure, split-and-pool metabarcoding for tens of thousands of particles, and in situ amplification of barcoded microbial rRNA for deep sequencing analysis. In addition, I introduce computational algorithms for microbial co-localization analysis and robust spatial association mapping, enabling the identification of fine-scale bacterial interaction patterns.