Bursaphelenchus trypophloei sp. n. is described from weakened and dead twigs of living or recently fallen aspen trees, Populus tremula, in Poland. All propagative stages of the nematode were present in larval galleries of a bark beetle, Trypophloeus asperatus, and its dauer juveniles could be found in the haemocoel of older larvae, pupae and adult beetles. Characteristic morphology of the male spicules with distinct cucullus, extended anterior vulval lip, lateral fields with four incisures, and number and arrangement of male caudal papillae indicate that B. trypophloei sp. n. is closely related to the xylophilus group. This relation has been further confirmed by DNA sequencing and phylogenetic analysis of the 28S and ITS-1 rDNA region. Bursaphelenchus trypophloei sp. n. can be separated from other species in the xylophilus group by the morphology of spicules which have a shorter capitulum and unique rostrum which is pointed somewhat anteriorly, relatively thick vulval flap, which is straight, parallel to the body long axis or bent towards the body wall at its distal end, and other morphological and morphometric characters, i.e., shape of female tail, position of excretory pore at or posterior to nerve ring, spicule length of 25.7 (23.1-28.0) μm (as measured along arc) and shape, moderate length, i.e., L = 702 (603-946) μm in females and L = 679 (543-828) μm in males, and slender body (a = 39.2 (33.0-45.2) and 40.9 (32.0-46.4) in female and male, respectively). The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). Full reproductive incompatibility of B. trypophloei sp. n. with B. xylophilus, B. mucronatus, B. fraudulentus, B. populi and B. doui has been demonstrated in in vitro diallelic cross-breeding. The newly described nematode revealed limited ability to develop and reproduce on Botrytis cinerea cultures although it produced large populations on laboratory cultures of Cytospora chrysosperma – a fungus naturally associated with galleries of the nematode vector, T. asperatus.