Seasonal shifts in bacterial diversity of microbial mats were analyzed in three hot springs (39–68 °C) of Patagonia, using culture-independent methods. Three major bacterial groups were detected in all springs: Phyla Cyanobacteria and Bacteroidetes, and Order Thermales. Proteobacteria, Acidobacteria and Green Non-Sulfur Bacteria were also detected in small amounts and only in some samples. Thermophilic filamentous heterocyst-containing Mastigocladus were dominant Cyanobacteria in Porcelana Hot Spring and Geyser, and Calothrix in Cahuelmó, followed by the filamentous non-heterocyst Leptolyngbya and Oscillatoria. Bacteroidetes were detected in a wide temperature range and their relative abundance increased with decreasing temperature in almost all samples. Two Meiothermus populations with different temperature optima were found. Overall, fingerprinting analysis with universal bacterial primers showed high similarities within each hot spring despite differences in temperature. On the other hand, Cahuelmó Hot Spring showed a lower resemblance among samples. Porcelana Hot Spring and Porcelana Geyser were rather similar to each other, possibly due to a common geological substrate given their geographic proximity. This was even more evident with specific cyanobacterial primers. The different geological substrate and the seawater influence in Cahuelmó might have caused the differences in the microbial community structure with the other two hot springs.