AbstractAlu sequences are present in humans in excess of 500 000 copies per haploid genome and represent the largest family of short interspersed repetitive elements (SINEs). These mobile genetic elements are ancestrally derived from the 7SL RNA gene and move throughout the genomes of primates by retroposition. Polymorphic Alu insertions have proven to be useful for population studies, paternity determinations and forensic applications. Additionally, a simple polymerase chain reaction (PCR)‐based assay has been established to examine these polymorphisms. In the present study, we have applied the technique of multiplex polymerase chain reaction to the Alu polymorphic system. Duplex and triplex PCR reactions were performed for the analysis of five different Alu polymorphic loci in different combinations. This study represents a starting point for further experimentation to improve and eventually optimize Alu multiplex PCR.