Simple SummaryMonitoring and maintaining genetic diversity is essential for the conservation of most species, including fallow deer. Various markers can be tested to estimate genetic diversity. For this purpose, microsatellites consisting of four-base-pair repetitive units are highly recommended due to their polymorphisms and comparable and reliable detection during analyses. In this study, nearly one hundred tetrameric microsatellites were collected from nine other deer species, which were tested on twenty individuals from five Hungarian fallow deer populations. As a result, 14 polymorphic markers were selected for the investigation panel, which fills a gap in the field of experts dealing with fallow deer genetics.AbstractThe fallow deer (Dama dama) represents significant game management value globally, and human activities are significantly impacting the species. Besides the positive effects, these activities can threaten its existence, health, and value. The aim of the authors was to develop a tetranucleotide microsatellite panel that could be clearly interpreted and used for genetic testing of fallow deer. Such a panel did not exist until now and could be particularly useful in the field of conservation genetics and forensics. A total of 99 tetrameric microsatellites, originally designed for related deer species, were tested on 20 fallow deer individuals from five Hungarian sampling areas. Original and newly designed primers were used to amplify the microsatellite regions using previously published or optimized PCR protocols. The lengths and sequences of specific amplicons were detected using capillary electrophoresis, and the rate of polymorphism was determined. Altogether, 80 markers provided PCR products of adequate quality and quantity. Among them, 15 markers proved to be polymorphic (2–5 alleles/locus), and 14 tetrameric markers were selected for further analysis. Statistical calculations showed that the selected polymorphic microsatellites can potentially enable key individualization in many areas of wildlife and population genetics, thus protecting the species.