This study presents the use of relaxed molecular clock methods to infer the dates of divergence between Panagrolaimus species. Autocorrelated relaxed tree methods, combined with well characterised fossil calibration dates, yield estimates of nematode divergence dates in accordance with the palaeontological age of fossil ascarid eggs and with the previously estimated date of 18 Ma (range 11.6 to 29.9 Ma) for the divergence of the Caenorhabditis lineage. Our data indicate that Panagrolaimus davidi from Antarctica separated ca 21.98 Ma from its currently known, most closely related strain. Thus, P. davidi may have existed in Antarctica prior to the Last Glacial Maximum, although this seems unlikely as it shares physiological and life history traits with closely related nematodes from temperate climates. These traits may have facilitated colonisation of Antarctica by P. davidi after the quaternary glaciation, analogous to the colonisation of Surtsey Island, Iceland, by P. superbus after its volcanic formation. This study demonstrates that autocorrelated relaxed tree methods combined with well characterised fossil calibration dates may be used as a method to estimate the divergence dates within nematodes in order to gain insight into their evolutionary history.