Microsatellites consist of short motifs (one to six nucleotides) that are repeated from four to 20+ times at points scattered across all eukaryotic genomes. They are useful as markers for genetic structure since the number of repeats at any given locus is unstable and new repeat variants are constantly arising by mutation and being lost by drift and other population-level events. Strategies to screen a total of 550 polymorphic microsatellite loci have been described in A. mellifera (Solignac et al., 2003), and many thousands more are found in the complete honey bee genome (Honey Bee Genome Sequencing Consortium, 2006). The protocol described here has been used to analyse the temporal genetic variation of island honey bee populations (Muñoz et al., 2012), the mating frequency of the Iberian honey bee (Hernández-García et al., 2009) and the population genetic structure of European honey bees (Muñoz et al., 2009; see also the BEEBOOK paper on characterizing subspecies and ecotypes by Meixner et al. (2013) for a full review of the use of microsatellites in determining honey bee ecotypes). It takes advantage of multiplexing, whereby multiple loci are screened in a single PCR reaction and size assay. These loci are widely used, and it is subsequently possible to compare allelic counts and genotypes across different studies.