Abstract A large number of reagents and steps are required for restriction fragment length polymorphism (RFLP) analysis, which at times make determining the cause of any observed anomaly difficult. Troubleshooting problems in RFLP analysis is difficult and often the exact cause of a problem cannot be determined. In this paper a collection of controlled experiments detail the consequences of a number of human or materials problems. Although the focus is on forensic applications, this troubleshooting guide will be helpful to anyone employing Southern analysis.