Background: When the motherʼs DNA profile is not available for paternity testing, there is a smaller probability that a locus will exclude an alleged father. This study aims to evaluate the risk of potential false paternity inclusions in motherless cases. Study Design and Methods: More than 20 000 duos were generated by removing the maternal genotypes from exclusion trios. After recalculating paternity in these duos, any found inclusions would be false. Results: The use of an appropriate number of loci, mutation model, and mutation rates to analyze motherless paternity cases was robust against false inclusions. A single potential false inclusion was observed in a case wherein kinship plays a role. This result highlights the importance of testing the mother when available and of obtaining information on family circumstances for the proper handling of cases involving related individuals. Conclusion: The guidelines we used here were sufficient to avoid false inclusions in a data set of more than 20 000 motherless cases.