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Cable bacteria (CB) perform electrogenic sulfur oxidation (e-SOx) by spatially separating redox half reactions over centimetre distances. For freshwater systems, the ecology of CB is not yet well understood, partly because they proved difficult to cultivate. This study introduces a new 'agar pillar' approach to selectively enrich and investigate CB populations. Within sediment columns, a central agar pillar is embedded, providing a sediment-free gradient system in equilibrium with the surrounding sediment. We incubated freshwater sediments from a streambed, a sulfidic lake and a hydrocarbon-polluted aquifer in such agar pillar columns. Microprofiling revealed typical patterns of e-SOx, such as the development of a suboxic zone and the establishment of electric potentials. The bacterial communities in the sediments and agar pillars were analysed over depth by PacBio near-full-length 16S rRNA gene amplicon sequencing, allowing for a precise phylogenetic placement of taxa detected. The selective niche of the agar pillar was preferentially colonized by CB related to Candidatus Electronema for surface water sediments, including several potentially novel species, but not for putative groundwater CB affiliated with Desulfurivibrio spp. The presence of CB was seemingly linked to co-enriched fermenters, hinting at a possible role of e-SOx populations as an electron sink for heterotrophic microbes. These findings add to our current understanding of the diversity and ecology of CB in freshwater systems, and to a discrimination of CB from surface and groundwater sediments. The agar pillar approach provides a new strategy that may facilitate the cultivation of redox gradient-dependent microorganisms, including previously unrecognized CB populations.Novel agar pillar gradient columns selectively enrich for cable bacteria and differentiate electrogenic populations from surface and groundwater sediments.