Mayaro virus (MAYV; genus Alphavirus, family Togaviridae) is a neglected tropical virus with distribution currently limited to Central and South America. Although MAYV infection can lead to a prolonged and debilitating polyarthralgia, there is limited surveillance and no approved antivirals or vaccines. Here, we compare the replication and transcription efficiencies of two MAYV strains with different geographic distributions, TRVL and BeAr, using a highly sensitive trans-replication system. In the trans-replication system, virus replicase proteins are uncoupled from the viral RNA template (i.e., the genomic strand). Initial experiments revealed that the replicase of the TRVL strain is more active in human cells, while the BeAr replicase is more active in mosquito cells. As mosquito and human cells are maintained at different temperatures, we investigated whether replicase activity is temperature-dependent. In human cells incubated at 28°C, TRVL replicase activity was more reduced than that of BeAr, indicating that TRVL replicase is more adapted to higher temperature conditions typical of the human host environment. Chimeric replicases with swapped nsP4, an RNA-dependent RNA polymerase, were constructed. The results with chimeric replicases indicated that if non-structural polyprotein P123 is derived from the BeAr strain, there is less efficient RNA replication in human cells, suggesting that determinants for different replicase activities do not reside within nsP4. Further, chimeric templates with exchanged 5’ UTRs between TRVL and BeAr were used to identify genomic regions responsible for different replicase activities. Replicases belonging to both strains generally replicated and transcribed their own, heterologous, and 5’ UTR-swapped template RNAs with similar efficiencies in human and mosquito cells. Furthermore, template cross-utilisation experiments demonstrated that both MAYV replicases can utilise template RNAs (i.e., genomic strand) of other alphaviruses, albeit with varying efficiencies. Additionally, replicases of other alphaviruses could utilise MAYV templates; however, efficiencies were substantially reliant on the cell type.