The transcriptional program of poxviruses is divided into early, intermediate and late phases enabled by a multisubunit DNA-dependent RNA polymerase and stage-specific transcription factors that recognize cognate promoters. Although promoter sequences are highly conserved among the different chordopoxvirus genera, the transcription factors exhibit considerable amino acid divergence that parallels the evolutionary distance of the host species. Thus, the large/small subunits of the intermediate transcription factors (ITFs) of salmon gill poxvirus, crocodilepox, canarypox, and myxoma have 23/29, 40/31, 51/38 and 58/65 % amino acid identity, respectively, to the vaccinia virus (VACV) orthologs. The purpose of the present study was to determine the functional interchangeability of the ITF subunits and their putative interactions with other elements of the transcriptional machinery. A quantitative readout of ITF function using firefly luciferase (Fluc) was obtained. The activity of the large subunit orthologs was greater than that of the small subunit orthologs, with both sets following the degree of sequence similarity in relation to VACV. The same pattern was obtained with both heterospecific (e.g., myxoma large and VACV small subunits) and homospecific (e.g., myxoma large and small subunits) pairings, suggesting inefficient interactions with other elements of the transcription system. When recombinant hybrid VACV expressing the Myxoma virus (MYXV) ortholog of the small subunit (A8) were blind passaged multiple times, their replicative abilities were enhanced. Complete genome sequencing of the virus populations revealed five mutations present in the two largest subunits of the viral RNA polymerase (RNAP) and two predicted expression-enhancing mutations around the translation initiation site of the MYXV A8 ortholog. Amplicon sequencing was used to quantify the frequency of each mutation in its respective population, which revealed that they increased as passaging occurred. This indicated a correlation with increased fitness, which then needed to be confirmed, so these mutations were all experimentally introduced into the original hybrid virus and demonstrated to enhance virus replication independently. These mutations were then characterized to determine their specific effects on the viral RNAP (vRNAP) and viral replication and transcription. This approach could have broader applications for studying essential genes in poxviruses and other viruses as well.