Actinobacteria are ubiquitous in soil, freshwater and marine ecosystems. Although various studies have focused on the microbial ecology of this phylum, data are scant on the ecology of actinobacteria endemic to hot springs. Here, we have investigated the molecular diversity of eubacteria, with specific focus on the actinobacteria in hot springs in Zambia, China, New Zealand and Kenya. Temperature and pH values at sampling sites ranged between 44.5 and 86.5 °C and 5-10, respectively. Non-metric multidimensional scaling analysis of 16S rRNA gene T-RFLP patterns showed that samples could be separated by geographical location. Multivariate analysis showed that actinobacterial community composition was best predicted by changes in pH and temperature, whereas temperature alone was the most important variable explaining differences in bacterial community structure. Using 16S rRNA gene libraries, 28 major actinobacterial OTUs were found. Both molecular techniques indicated that many of the actinobacterial phylotypes were unique and exclusive to the respective sample. Collectively, these results support the view that both actinobacterial diversity and endemism are high in hot spring ecosystems.