The length polymorphism of nuclear microsatellite is one of the important molecular markers for landscape genetics and phylogeography studies. Microsatellites with proper polymorphism and an explicit mutation mechanism are critical for the accuracy of research results. We adopted 15 widely used nuclear microsatellites for Fraxinus to F. mandshurica to investigate whether the primers could stably work in these species. By using FIASCO, we developed 9 nuclear microsatellite primers for F. mandshurica. To analyze the mutation mechanism of length variation, also we sequenced the PCR products. We found that only 66.7% of primers for Fraxinus could work in F. mandshurica. Among these, 70% were reported to have an insert/deletion in flanking regions. For the 9 newly developed nuclear microsatellites, 33.3% closely size homoplasy. Our study revealed that size homoplasy is common in microsatellites, especially for those closely related species.