<i>Background:</i> The dust mite <i>Lepidoglyphus destructor</i> is a major source of mite allergy in European rural environments, but it also causes allergy in urban populations around the world. We have previously cloned, sequenced and expressed several allergens from <i>L. destructor</i> (Lep d 2, Lep d 5, Lep d 7 and Lep d 13). The aim of this study was to identify and clone additional allergens from <i>L. destructor</i>, and to evaluate their IgE-binding reactivities. <i>Methods:</i> PCR and screening with sera from <i>L. destructor</i>-sensitised individuals were used to isolate new clones from a phage display <i>L. destructor</i> cDNA library. The complete coding sequences of the clones were determined and expressed as His₆-tagged recombinant proteins in <i>Escherichia coli</i>. The recombinant proteins were analysed by SDS-PAGE, immunoblotting and mass spectrometry. <i>Results:</i> Two new clones, showing homology to tropomyosin and α-tubulin in several species, were isolated from the phage display <i>L. destructor</i> cDNA library. Due to its homology to group 10 dust mite allergens, the tropomyosin clone was named Lep d 10. The IgE-binding frequencies of the recombinant Lep d 10 and α-tubulin were 13% (18/136) and 12% (11/95), respectively, among subjects with IgE reactivity to mites and/or crustaceans. <i>Conclusions:</i> Two new allergens from <i>L. destructor</i> have been identified and can now be added to the repertoire of recombinant <i>L. destructor</i> allergens. In addition, both these allergens belong to highly conserved protein families and may be important for evaluation of allergenic cross-reactivity.