Objective: A set of microsatellite markers with high polymorphism from Tsaiya duck were used for the genetic monitoring and genetic structure analysis of Brown and White Tsaiya duck populations in Taiwan.Methods: The synthetic short tandem repeated probes were used to isolate new microsatellite markers from the genomic DNA of Tsaiya ducks. Eight populations, a total of 566 samples, sourced from Ilan Branch, Livestock Research Institute were genotyped through novel and known markers. The population genetic variables were calculated using optional programs in order to describe and monitor the genetic variability and the genetic structures of these Tsaiya duck populations.Results: In total 24 primer pairs, including 17 novel microsatellite loci from this study and seven previously known loci, were constructed for the detection of genetic variations in duck populations. The average values for the allele number, the effective number of alleles, the observed heterozygosity, the expected heterozygosity, and the polymorphism information content were 11.29, 5.370, 0.591, 0.746, and 0.708, respectively. The results of analysis of molecular variance and principal component analysis indicated a contracting Brown Tsaiya duck cluster and a spreading White Tsaiya duck cluster. The Brown Tsaiya ducks and the White Tsaiya ducks with Pekin ducks were just split to six clusters and three clusters when K was set equal to 6 and 3 in the Bayesian cluster analysis. The individual phylogenetic tree revealed eight taxa, and each individual was assigned to its own population.Conclusion: According to our study, the 24 novel microsatellite markers exhibited a high capacity to analyze relationships of inter- and intra-population in those populations with a relatively limited degree of genetic diversity. We suggest that duck farms in Taiwan could use the new (novel) microsatellite set to monitor the genetic characteristics and structures of their Tsaiya duck populations at various intervals in order to ensure quality breeding and conservation strategies.