Premise: Progeny of avocado (Persea americana) are highly variable due to high levels of heterozygosity. Breeding programs need molecular resources to allow the assessment of genetic differences and the selection of genotypes. Polymorphisms that uniquely identify different avocado cultivars provide a valuable tool to accelerate avocado research and development, including, for example, genotype selection. Methods: A double-digest restriction site–associated DNA sequencing (ddRADseq) approach was used to screen 10 avocado cultivars for single-nucleotide polymorphisms (SNPs). The fragments were size selected with Blue Pippin and PCR using universal Illumina primers, and catalog tags were then created with de novo alignment using Stacks software. Catalog tags were tabulated and filtered to identify alleles unique to each cultivar. Results: A total of 104 million sequences were collected, and 52 homozygous SNPs were identified that uniquely distinguished nine avocado cultivars. The cultivars Carmen Hass and Hass have a strong genetic similarity and no homozygous SNPs distinguishing these cultivars could be identified; therefore, both cultivars were grouped together. Discussion: The resource described here for cultivars of P. americana presents a new and significant molecular resource that can enable targeted genotype selection, paternity analysis, germplasm genotyping, pollination dynamics investigation, and crop improvement.