We have characterized RSHb, a hemoglobin cDNA clone isolated from a seedling cDNA library of the small radish (Raphanus sativus L. var. sativus). Its 480-bp ORF encodes a protein of 160 amino acids, with a predicted molecular weight of 17.98 kDa and an estimated pi of 8.77. Sequence analysis and multiple alignment revealed that the RSHb shares 95% nucleotide sequence and 93% amino acid sequence identities with those of AHB 1 from Arabidopsis thaliana, a Class I non-symbiotic hemoglobin. Our Southern blot analysis showed that RSHb is a single-copy gene in the small radish genome. The northern blot analysis demonstrated that this gene is expressed in all organs, including the roots, hypocotyls, and leaves, with transcription levels increasing under flooding and high-sucrose conditions. However, cold treatment does not affect its expression pattern. We conclude that RSHb is a Class I non-symbiotic hemoglobin gene.