Autosomal short tandem repeat (STR) markers remain the cornerstone of modern forensic genetics, providing exceptional power for individualization, kinship verification, and reconstruction of complex investigative cases. Over the last decade, the field has undergone a major technological transition from length-based capillary electrophoresis (CE) toward sequence-level characterization using massively parallel sequencing (MPS), enabling detection of internal sequence variants (isoalleles) and flanking-region polymorphisms that substantially increase discriminatory power in many forensic contexts. Although MPS is increasingly adopted in forensic laboratories, implementation remains dependent on infrastructure, cost considerations, validation requirements, and jurisdiction-specific legal frameworks. This review synthesizes the molecular mechanisms underlying STR variability, including replication slippage and mutation processes, and critically evaluates the transition to sequencing-based analysis. Particular attention is given to analytical challenges such as stochastic effects in ultra-low-template DNA and PCR inhibition in degraded samples. Special emphasis is placed on identification of skeletal remains from mass graves and historical contexts, where hierarchical analytical strategies—from mini-STR approaches to MPS-based workflows—enable recovery of highly fragmented DNA. The review also examines the evolution of probabilistic genotyping (PG), highlighting the importance of algorithmic transparency and reproducible analytical frameworks for judicial applications. By integrating technological advances with practical forensic challenges, this review outlines a comprehensive framework for implementing high-resolution STR analysis in contemporary genomic casework. As a narrative synthesis, the conclusions reflect currently available published evidence and acknowledge variability in validation status, implementation practices, and regional forensic infrastructures.
Irena Zupanič Pajnič (Fri,) studied this question.