Cacao (Theobroma cacao L.) is an important cash crop in many tropical countries. Cacao accessions must be propagated vegetatively to conserve genetic integrity due to its allogamous nature and its seed recalcitrance (lack of dormancy). Therefore, cacao germplasm is usually maintained as living trees in field collections and has resulted in varying rates of misidentification and duplication. Using a high throughput genotyping system with 15 microsatellite loci, all 924 trees in the USDA‐ARS Mayaguez cacao collection were fingerprinted. Nineteen accessions (12.3%) were found to have intraplant errors while 14 (9.1%) synonymous sets were identified that included replicates of 49 accessions. The average number of alleles (8.8; SE = 0.56) and gene diversity (HObs = 0.65; SE = 0.026) indicate a high allelic diversity in this collection. A distance‐based cluster analysis and a Bayesian assignment test showed that the cacao accessions can be classified into four distinct clusters, with their geographical origins covering most of the cacao growing regions in the Americas. Assessment of the representative diversity of the collection led to the identification of several genetic gaps, including underrepresented genetic populations and particular traits of economic and agronomic value. The improved understanding of identities and structure in the USDA‐ARS cacao collection will contribute to more efficient use of cacao in conservation and breeding.

Microsatellite-based DNA fingerprinting has been increasingly applied to cacao (Theobroma cacao L.) genotype identification. However, the accuracy and reliability of using high throughput microsatellite analysis for cacao clone identification have not yet been rigorously assessed. Despite the use of highly robust fingerprinting protocols, cacao genotype identification has been affected by genotyping errors, which potentially mislead the result of clone identification. In this paper, we calculated the probability of identity for 15 selected microsatellite loci. We then quantified the genotyping error rate through repeated genotyping and simulated the impact of the genotyping error on cacao clone identification. Allelic dropout (ADO), or failure to amplify one allele for a heterozygous locus, and false allele (FA), or an amplicon size error by the polymerase, accounted for 48 and 52% of the genotyping inconsistencies, respectively. The result of simulation showed that 99% of the consensus genotype can be generated for the ambiguous loci through a minimum of three polymerase chain reaction (PCR) repetitions. On the basis of the error rate and probability of identity (PID), we designed a genotyping scheme and applied it to the cacao germplasm held in the USDA cacao collection at Mayaguez, Puerto Rico. Out of the 141 samples, we unambiguously identified nine duplicated groups consisting of 34 cacao accessions. This genotyping scheme is being implemented in large scale fingerprinting of cacao germplasm.

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