Free-living Actinobacteria are universally recognized as high-GC organisms. Freshwater Actinobacteria have been identified as abundant and prevalent members of freshwater microbial communities, but the two most common lineages (acI and acIV) have remained impossible to culture to date. We have analysed metagenomic data from lakes and estuaries, and show that members of acI and acIV are indeed abundant. We then show that the majority of actinobacterial reads from metagenomic datasets (both lakes and estuaries) are consistently low GC. Analysis of assembled scaffolds from these datasets also confirms that actinobacterial scaffolds are primarily low GC, although high-GC scaffolds were also observed, indicating both types of Actinobacteria coinhabit. Phylogenetic analysis of 16S rRNA gene sequences, both from PCR-based clone libraries and metagenomic reads, and the discovery of a low-GC scaffold containing a partial 16S rRNA gene, points to the abundance of the well-known acI and acIV lineages of freshwater in these habitats, both of which appear to be low GC.