The microneme protein-1 (MP-1) of Plasmodium knowlesi and Plasmodium vivax facilitates merozoite invasion of the erythrocyte by binding to Duffy blood group antigens. Since this protein is important in the invasion process and is a potential vaccine candidate, it is important to understand the nature of diversity within the MP-1 gene. Nine MP-1 gene sequences were compared from 2 isolates of P. knowlesi and a laboratory strain of P. vivax. The MP-1 genes of P. knowlesi were dimorphic based upon the central hydrophilic regions (III and IV) that were well conserved as alpha and beta types. Other regions were conserved among all P. knowlesi genes except for the amino cysteine-rich region (region II), a region predicted to be the initial contact site of the erythrocyte binding domain. Two distinct sequence motifs and part of a third were identified in region II that had a common identity of 68%. In some MP-1 genes recombination had occurred to create hybrids of the two sequence types. All cysteines and aromatic amino acids of region II were conserved in all genes or within a sequence type. There were 2 apparent recombination points within region II where switching occurred between sequence types. Another possible recombination site, identified as a common sequence motif, was identified in the middle of the hydrophilic region, at the beginning of regions III or IV. Nonsynonymous mutations within region II were biased towards radical amino acid changes, especially towards the carboxyl third, where there were 3 distinct types of sequence. Most synonymous and nonsynonymous nucleotide mutations within regions I, V, and VI were infrequent, individual events and not associated with any particular sequence type. Cysteine-rich regions of the P. vivax MP-1 gene compared to the P. knowlesi genes were characterized by an increased number of synonymous and nonsynonymous changes. This data identifies 2 mechanisms for generation of diversity in the MP-1 gene family, intergenic recombination and nucleotide mutations. Both may be mechanisms the parasite uses to evade the host immune response or to alter erythrocyte receptor specificity.