The first documentation of facultative parthenogenesis (FP) in non-avian reptiles (snakes) occurred in 1997, following the application of molecular genetic methods for parentage analysis, and since has been described in only four additional snake species. Here, in two species of live-bearing (viviparous) North American pitviper snakes, using microsatellite DNA fingerprinting, we describe the first record of a virgin birth by FP in the copperhead (Agkistrodon contortrix), and the first genetically confirmed case of long-term sperm storage (LTSS) of exceptional duration (5 years) in the eastern diamond-backed rattlesnake (Crotalus adamanteus). The capacity of female squamate reptiles (snakes, lizards, and amphisbaenians) to store viable sperm for extended periods of time is a competing hypothesis to FP, and it is often inferred when interpreting certain births where adult females have been isolated from adult male mates for prolonged periods. On reviewing the literature of certain cases of LTSS in snakes, we provide compelling support for the prevalence of FP, casting doubt over the widespread acceptance of LTSS. Accordingly, with the discovery of FP in squamates and the results of the present study, we advocate that in order to differentiate between LTSS and FP, especially under natural conditions, rigorous molecular testing will be required. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104, 934–942.