Myosin heavy chain DNA sequence data is tested for the first time for resolving phylogenetic relationships within different levels of orb-weaving spiders, from family through species level variation. Primers were designed that span a variable length intron in the myosin II heavy chain gene. Variable length (550-721 bp) sequences were obtained from 44 taxa of spiders. Monophyly of the orb weaving family Araneidae was tested using 36 ingroup genera, two additional families of orb weaving spiders Ð Tetragnathidae (which spin traditional orbs) and Linyphiidae (which spin modified sheet webs), and taxa in the non-orb weaving families Amaurobiidae and Pisauridae. A generic level phylogeny for the Araneidae was produced to test the ability of myhc II to recover monophyletic genera and relationships that were strongly supported in previous morphological studies. Finally, a species level phylogeny of mastophorine and cyrtarachnine spiders was produced to test the behavioral hypothesis that specialized bolas web hunting in Mastophora and Ordgarius evolved through a gradual specialization of orb webs in Cyrtarachne, Poecilopachys, and Pasilobus.