Few ophiostomatoid fungi have been reported from the margin of the Tibetan Plateau and none have been found in the central portion of the region. In a survey of ophiostomatoid fungi associated with spruce bark beetles in Tibetan subalpine forests, numerous strains of Leptographium s. l. (Ophiostomataceae) were isolated from Orthotomicus sp. (Coleoptera: Scolytinae) and its galleries infesting Picea likiangensis var. balfouriana. Morphological characters and phylogenetic analysis based on multiple DNA sequence data (ITS2-partial LSU rDNA region, beta-tubulin and transcription elongation factor-1α genes) revealed a new species in the “Grosmannia penicillata complex”, which is proposed as G. tibetensis. The species is characterized by both Leptographium and Pesotum asexual states, which is unique in the “G. penicillata complex”. Additionally, sequences of the tubC paralogue gene were found combining with tub2 sequences in many species of the “G. penicillata complex”, resulting in incongruent trees. This is the first report of tubulin paralogue genes in ophiostomatoid fungi. Gene duplication and losses make beta-tubulin a potentially challenging locus for use as a molecular marker for tracing speciation.