The timber rattlesnake (Crotalus horridus) occurs in discontinuous populations across the eastern and central United States. The species exhibits high levels of polymorphism in morphological traits, especially in coloration and pattern. Previous studies recognized either distinct northern and southern subspecies or three regional morphs (northern, southern, and western), but the conflicting data sets and limited geographic sampling of previous studies have left the relationships among those regional variants unclear. In this study, univariate and multivariate statistics, analyzed in conjunction with a geographic information system, were used to examine geographic variation in 36 morphological characters recorded from 2420 specimens of C. horridus across its range.

Univariate analyses detected substantial geographic variation in all meristic characters. Scutellation exhibited a general north–south pattern of variation, and most scale counts averaged higher in southern regions. Pattern characters differentiated the northeastern, central-eastern, and north-central regions from the southern and western regions. Coloration displayed a pattern of strong clinal variation among three broad areas consisting of the combined northeastern, central-eastern, and southern Appalachian regions, the northwestern regions, and the southern regions. Morphometric characters exhibited a general north–south pattern of geographic variation, with larger head and body sizes in southern regions. Sexual dimorphism was strong in ventral and subcaudal scales, and weak to moderate in band length and band spacing.

Principal component analysis indicated that band length was the most important variable for characterizing geographic variation. The northeastern regions remained moderately distinct in all multivariate cluster analyses. The northwestern regions appeared very distinct in most cluster analyses for females. However, the clusters in all models showed extensive geographic overlap. Maps of the clusters revealed two north–south patterns of clinal variation across the northeastern, northwestern, and southern regions. The discordant patterns of variation among individual characters, overlapping patterns of coloration, and extensive overlap among the multivariate clusters collectively indicate that the putative zones of intergradation among recognized subpopulations of C. horridus are much broader than previously thought. Furthermore, because the general patterns of geographic variation are strongly clinal, our results support the conclusion that C. horridus is a single widespread species with variation too extensive and complex to be reflected by formal subspecific designations.