Microsatellite primers were developed for the evergreen tree Laurus to investigate population genetic structure and patterns of gene flow via animal-dispersed pollen and seeds. Twenty polymorphic nuclear microsatellite markers were developed using CA, GA, AAC, and ATG n-enriched genomic libraries. Given the tetraploidy of the sampled populations, we analyzed our data both as dominant loci and as codominant genotypic data to calculate allele frequencies and genetic diversity. A total of 196 and 222 alleles were found in 37 Mediterranean (L. nobilis) and 26 Macaronesian islands (L. azorica) individuals, respectively. Levels of polymorphism of the reported markers are adequate for studies of diversity and parentage in natural populations of this Tertiary relict tree.