Background: Paternity investigations play an important role in determining biological relatedness, and in South Africa, the outcome of these investigations impacts medical, judicial and home affairs decisions. Short Tandem Repeat (STR) analysis is utilised to perform paternity and kinship analysis, due to the polymorphic nature of STR loci. The cost associated with paternity testing is high, and there is a demand for motherless testing. Objectives: This study aims to determine what the impact of motherless testing would have been by evaluating 6182 pa- ternity trio cases. Methods: The AmpFLSTR™ Identifiler™ PCR Amplification kit was used to profile each of the trio cases. A scenario was created where the mother was eliminated from the test results to determine if the paternity outcome would change. Results: Putative fathers were excluded in 27% of all cases, and in 2.5% of those cases, putative fathers would have been falsely included, had the mother not been tested. These false inclusions are attributed to coincidental STR loci that are shared between the mother and the putative father. The addition of loci to the STR profiling kit may resolve the issue; however, comparable STR data with more loci will have to be evaluated to ensure it overcomes the issue of coincidentally shared loci between unrelated individuals. Conclusion: We would recommend that within our setup and within similar setups, the mother always be included for test- ing, except in extreme scenarios such as death. False inclusion of putative fathers could have serious legal implications for testing laboratories. Keywords: Motherless paternity testing; South African population.
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