Syntrophy and mutualism play a central role in carbon and nutrient cycling by microorganisms. Yet our ability to recognize these partnerships in nature or to effectively study their behavior in culture has been hindered by the inherent interdependence of syntrophic associations, their dynamic behavior, and their frequent existence at thermodynamic limits. Now solutions to these challenges are emerging in new methodologies. These include: comparative metagenomics and transcriptomics; discovery-based methods such as Magneto-FISH; and metabolic substrate tracking using stable isotopes coupled either with density gradient separation (SIP) or with FISH-SIMS. These novel approaches are redefining the way we study microbial mutualism and are making intimate microbial associations accessible to both identification and characterization in their native habitats.