This study investigated the genetic diversity and population structure of C. hoffmanni sloths in Costa Rica, revealing high genetic diversity and almost nonexistent population structuring. The study used Bayesian clustering analysis to determine the most likely number of genetic groups. The Northern group was the most genetically divergent, with high average homozygosity by locus, indicating higher levels of inbreeding. The South-East grouping was more genetically diverse, with high levels of admixture and lack of population structuring. The long-term effects of translocations of individuals from their area of origin into the South-East region are unknown. The study provides baseline information on existing sloth genotypes and provides evidence that sloths are not being returned to their point of origin. Further in-depth analyses are required to determine the extent of barriers and drivers on the genetic structure of sloths in Costa Rica. Anthropogenic pressures on sloth populations in Costa Rica are increasing, and further research is needed to investigate the genetic diversity of highly structured populations and determine the fitness of populations isolated through urbanization.