Researchers have long debated which genomic estimator of relatedness best captures the degree of relationship between two individuals. In the genomics era, this debate continues, with relatedness estimates being sensitive to the method used to generate genomic markers (e.g., reduced-representation sequencing, whole genome resequencing), marker quality, and levels of diversity in sampled individuals.Here, we compare six commonly used relatedness estimators (kinship genetic distance (KGD), Wang Maximum Likelihood (TrioML), Queller and Goodnight (Rxy), KING-robust, RAB, allele-sharing co-ancestry) across five species bred in captivity–including three birds and two mammals–with varying degrees of reliable pedigree data, using reduced-representation and whole genome resequencing data.Relatedness estimates varied widely across estimators, sequencing method, and species, yet the most consistent results with known pedigree data were found using KING-robust and to a lesser extent KGD. The allele-sharing estimator was sensitive to missing data and inbreeding, attributes that make this estimator ill-suited for use in captive breeding programs. Our combined results indicate there is not a single genomic based estimator that is ideal across different species and data types.To enable researchers to evaluate the most appropriate relatedness estimator for each new data set, we provide a structured workflow that is broadly applicable to conservation breeding programs, particularly where genomic estimates of relatedness can complement and complete poorly pedigreed populations. Given a growing interest in wild pedigrees, our results and workflow are also applicable to in situ wildlife management.

No abstract