This study presents the isolation and characterization of a novel nonsymbiotic Hb gene from sessile oak (Quercus petraea) seedlings, herein designated QpHb1. • The cellular and tissue expression of QpHb1 was analysed by Northern blotting and in situ hybridization. • The encoded protein was predicted to consist of 161 amino acid residues, and shares 71 and 51% amino acid sequence identity with the Arabidopsis class 1 and 2 nonsymbiotic Hb, respectively. Northern blot analysis revealed that QpHb1 was strongly expressed in roots. Spatial expression analysis of QpHb1 in the root apical region of sessile oak by in situ hybridization indicated that transcripts were mostly abundant in protoxylem cell initials, some cortical cells and the protoderm. In addition, when comparing the expression profile of QpHb1 in sessile and pedunculate oak (Quercus robur), two species with contrasted hypoxia tolerance, the transcript level of QpHb1 rose early in the most flood-tolerant species, pedunculate oak, during root submergence. • The spatial–temporal expression of QpHb1 suggests that this gene could participate in perception and signalling during hypoxia.