Plasmodium falciparum parasites use multiple ligand-receptor interactions to invade human erythrocytes. Variant expression levels of members of the PfRh and PfEBA ligand families are associated with the use of different erythrocyte receptors, defining invasion pathways. Here we analyse a major polymorphism, a large sequence deletion in the PfRh2b ligand, and erythrocyte invasion profiles in uncultured Senegalese isolates. Parasites vary considerably in their use of sialic acid-containing and protease-sensitive erythrocyte receptors for invasion. The erythrocyte selectivity index was not related to invasion pathway usage, while parasite multiplication rate was associated with enhanced use of a trypsin-resistant invasion pathway. PfRh2b protein was expressed in all parasite isolates, although the PfRh2b deletion was present in a subset (approximately 68%). Parasites with the PfRh2b deletion were found to preferentially utilize protease-resistant pathways for erythrocyte invasion. Sialic acid-independent invasion is reduced in parasites with the PfRh2b deletion, but only in isolates derived from blood group O patients. Our results suggest a significant role for PfRh2b sequence polymorphism in discriminating between alternative erythrocyte receptors for invasion and as a possible determinant of virulence.