Differential screening of an alfalfa root nodule cDNA library with either root or nodule mRNA resulted in the isolation of two groups of leghemoglobin cDNA which differ significantly in sequence. Analysis of one member of each group revealed a divergence within the coding region of 15% at the nucleotide level and 14% at the amino acid level. The 3′ non-coding sequences are 25% divergent but are highly conserved over a stretch of 54 nucleotides which contains two sequence motifs common to leghemoglobin genes from other plant species. Southern blotting analysis with exon-specific probes has shown that there are approximately twice as many leghemoglobin gene copies in the alfalfa genome corresponding to one type of cDNA as compared with the other. Using the same criterium of DNA sequence relatedness these two distinct groups of leghemoglobin genes have also been identified in the genomes of the diploid annual Medicago truncatula and the closely related genus, Melilotus. Transcripts corresponding to both groups of leghemoglobin genes are first detected in alfalfa nodules 9–10 days after Rhizobium inoculation. Thereafter, mRNA levels increase rapidly and synchronously, reaching a maximum approximately 2 days later. There is a 2–3 fold difference in the steady-state levels of the two mRNA populations and this is maintained throughout the subsequent two weeks of nodule growth. The absence of any detectable transcription during the early stages of nodule development and the apparent co-ordinate expression of leghemoglobin genes in alfalfa contrasts with the situation in soybean and suggests that important differences in leghemoglobin gene regulation exist between these two distantly related legume species.