Feijoa [Acca sellowaiana (O. Berg) Burret], also known as pineapple guava or goiabeira‐serrana, is a species of the Myrtaceae family native to southern Brazil and Uruguay. The genetic diversity of feijoa's natural populations has been studied and is relatively well‐documented; however, the genetic diversity of feijoa accessions in the largest active germplasm bank (AGB) in Brazil, and is still poorly documented. Characterizing and conserving pineapple guava accessions present in the AGB is essential for developing breeding programs and conserving the diversity of the species. Thus, the objective of this study was to characterize the genetic diversity of all accessions using eight microsatellite markers. The AGB collection includes accessions from 26 municipalities in Brazil, New Zealand, the United States, Israel, and Uruguay; selected clones, and selections of directed crossings, totaling 313 accessions. Genotyping 309 of these accessions with eight microsatellite markers revealed 119 alleles. Polymorphism information content (PIC) values varied between .466 (ASE40) and .890 (ASE34) with an overall mean of .718. This genotyping confirms the existence of two main types or biotypes in the species, denoted as the Uruguay type and Brazil type. Also, a significant proportion of the feijoa diversity, which is absent from the AGB‐Feijoa collection, is present in germplasm maintained by indigenous people, quilombola communities, federal conservation units, participatory genetic breeding, and areas of natural occurrence, thus reinforcing the need to introduce new accessions into São Joaquim's AGB‐Feijoa.