This study examines the population structure of Classic period (A.D. 250–900) Maya populations through analysis of odontometric variation of 827 skeletons from 12 archaeological sites in Mexico, Guatemala, Belize, and Honduras. The hypothesis that isolation by distance characterized Classic period Maya population structure is tested using Relethford and Blangero's (Hum Biol 62 (1990) 5–25) approach to R matrix analysis for quantitative traits. These results provide important biological data for understanding ancient Maya population history, particularly the effects of the competing Tikal and Calakmul hegemonies on patterns of lowland Maya site interaction. An overall FST of 0.018 is found for the Maya area, indicating little among-group variation for the Classic Maya sites tested. Principal coordinates plots derived from the R matrix analysis show little regional patterning in the data, though the geographic outliers of Kaminaljuyu and a pooled Pacific Coast sample did not cluster with the lowland Maya sites. Mantel tests comparing the biological distance matrix to a geographic distance matrix found no association between genetic and geographic distance. In the Relethford–Blangero analysis, most sites possess negative or near-zero residuals, indicating minimal extraregional gene flow. The exceptions were Barton Ramie, Kaminaljuyu, and Seibal. A scaled R matrix analysis clarifies that genetic drift is a consideration for understanding Classic Maya population structure. All results indicate that isolation by distance does not describe Classic period Maya population structure. Am J Phys Anthropol, 2007. © 2006 Wiley-Liss, Inc.