The coefficient of coancestry (fAB) between individuals A and B is the classical measure of genetic relationship. fAB is determined from pedigree records and is the probability that random alleles at the same locus in A and B are copies of the same ancestral allele or identical by descent (ibd). Recently, the proportion of molecular marker variants shared between A and B (SAB) has been used to measure genetic relationship. But SAB is an upwardly-biased estimator of fAB, especially between distantly-related lines. fAB, SAB, and adjusted (to remove bias) estimates of molecular marker similarity (f AB (M) ) were compared. RFLP banding patterns at 46 probe-restriction enzyme combinations were obtained for 23 maize inbred lines derived from the Iowa Stiff Stalk Synthetic (BSSS) maize (Zea mays L.) population, and for 4 non-BSSS lines. f AB (M) was estimated as [Formula: see text], where Ξ΄ A (or Ξ΄ B) was the average proportion of RFLP variants shared between inbred A (or inbred B) and the non-BSSS lines. The average fAB among 253 pairwise combinations of BSSS lines was 0.212, whereas the average SAB was 0.397. The average f AB (M) was 0.162, indicating that the upward bias in SAB was effectively removed. SAB and fAB were significantly different (Ξ± = 0.05) in 76.3% of the comparisons, whereas 24.9% of the f AB (M) values differed significantly from fAB. The latter result suggests that selection and/or drift were present during inbred line development and that fAB may not be an accurate measure of the true proportion of ibd alleles between two lines. Cluster analyses based on S AB (M) and f AB (M) grouped lines according to pedigree, although several exceptions were noted. The presence of shared molecular marker variants between unrelated lines must be considered when setting SAB-based minimum distances for varietal protection. Under simplified conditions, more than 250 molecular marker loci are necessary to obtain sufficiently precise estimates of coefficient of coancestry using molecular markers.