Background <p>Polymerase chain reaction (PCR) applications including sequencing rely on thermostable DNA polymerases and their ability to generate accurate amplicons. Polymerization errors may hinder the detection of low-level DNA variants such as mutations in clinical samples or DNA from minor contributors in crime scene traces with DNA from multiple individuals. Short Tandem Repeat (STR) markers are affected by both random base substitutions and stutter, i.e., products which have lost or gained repeat units. The mechanisms leading to stutter formation have not yet been fully elucidated.</p> Results <p>Here, we applied an STR assay based on Unique Molecular Identifiers to study the effects of DNA polymerases with different characteristics on amplicon yield and formation of PCR errors. The levels of base substitutions were clearly connected to the fidelity of the DNA polymerases, which in turn was coupled with having an integrated 3’ to 5’ exonuclease domain. Stutter formation was not associated with fidelity. DNA-binding domains improve processivity, which in turn has been suggested to lower the incidence of stutter. However, no such effect was seen in the present study as a polymerase having a DNA-binding domain gave the highest stutter levels.</p> Conclusions <p>Overall, the degree of stuttering is likely due to several different DNA polymerase characteristics affecting the stability of the ternary complex and extension kinetics. This study highlights the importance of an increased understanding of DNA polymerase function and how this can influence the quality of the sequencing results, especially when analyzing complex parts of the human genome such as STR markers.</p> Graphical abstract <p></p>