Knowledge of the genetic relatedness between individuals is important in many research areas in quantitative genetics, conservation genetics, forensics, evolution, and ecology. In the absence of pedigree records, relatedness can be estimated from genetic marker data using a number of estimators. These estimators, however, make the critical assumption of a large random mating population without genetic structures. The assumption is frequently violated in the real world where geographic/social structures or nonrandom mating usually lead to genetic structures. In this study, I investigated two approaches to the estimation of relatedness between a pair of individuals from a subpopulation due to recent common ancestors (i.e., relatedness is defined and measured with the current focal subpopulation as reference). The indirect approach uses the allele frequencies of the entire population with and without accounting for the population structure, and the direct approach uses the allele frequencies of the current focal subpopulation. I found by simulations that currently widely applied relatedness estimators are upwardly biased under the indirect approach, but can be modified to become unbiased and more accurate by using Wright's F st to account for population structures. However, the modified unbiased estimators under the indirect approach are clearly inferior to the unmodified original estimators under the direct approach, even when small samples are used in estimating both allele frequencies and relatedness.K NOWING the degree of relatedness between individuals is essential in many research areas in quantitative genetics, conservation genetics, forensics, evolution, and ecology (Ritland 1996;Lynch and Ritland 1999;Weir et al. 2006). The expected value of relatedness between two individuals (e.g., 0.5 for parent-offspring in a large random mating population) can be easily calculated from their pedigree records. When pedigree is unavailable, incomplete, or unreliable, genetic marker information can be used instead to obtain an estimate of the realized value of relatedness, using a number of estimators developed for this purpose (e.g