We were interested in tracing the evolutionary paths leading to the development of thermostable extant enzymes, such as P450-TT; many ASR studies using sequences from mesophiles and one thermophilic family of proteins have reported increases in thermostability in deeper ancestors, but it was not clear if the same would be true for enzymes from thermophiles within a larger, predominantly mesophilic tree . In our studies we found a range of stability profiles for the haem domain, however, no clear overall trends were observed across the tree, and the oldest ancestors were not the most thermostable. Overall, there appeared to be considerable variability in the 15 T50 values of ancestors of various lineages, with intermediate nodes varying in 15 T50 between ~ 42 and 51 °C. However, ignoring the most basal node of the tree, N0, which joins the proteobacterial clade with the A205-F206 Diversity in the CYP116B Family actinobacterial outgroup so cannot be strictly considered as a potential historical ancestor, a general trend towards gain of thermostability over time can be seen in lineages terminating in a thermostable extant form (i.e., P450-AX, -TT, or -TB). For example, in the P450-TT lineage, for which more intermediate nodes were characterised, the ancestors closest to P450 TT were also thermostable, with both N115 and N106 having a 15 T50 of 50 °C, suggesting a trend towards gain of thermostability in this lineage. These differences in thermostability may reflect a transition from meso-to thermophilic bacteria in some lineages, e.g., leading to Tepidiphilus thermophilus , where there would have been selection pressure for enhanced thermostability. Characterisation of additional intermediate or descendant nodes in other lineages would allow better definition of trends in other branches of the tree, including the point at which thermostability arose in different CYP116B lineages. It would be of particular interest to assess descendants of N3 and N23, which showed relatively high 15 T50 values, and determine whether any of the uncharacterised source organisms of their descendants are thermophiles.