Plasmodium falciparum variant surface antigens (VSA) are targets of protective immunity to malaria. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and repetitive interspersed family (RIFIN) proteins are encoded by the two variable multigene families, var and rif genes, respectively. Whereas PfEMP1s are known to mediate cytoadhesion, the function of RIFINs is unknown. The sequence diversity and organisation of rif genes of the P. falciparum clones 3D7, HB3, DD2, and IT/FCR3 were investigated using a tree-building method which allowed sub-grouping of RIFINs into distinct groups. Two novel rif gene groups, rifA1 and rifA2, containing inter-genomic conserved rif genes, were identified. All rifA1 genes were orientated head-to-head with a neighbouring Group A var gene whereas rifA2 was present in all parasite genomes as a single copy gene with a unique 5' untranslated region. Rif transcript levels were determined in two different parasite lines, 3D7-Lib and NF54-VAR2CSA, expressing VSA associated with severe malaria in children and pregnant women, respectively. The 3D7-Lib showed high transcript levels of Group A var and neighbouring rif genes, whereas rifA2 was found highly transcribed in the VAR2CSA-expressing parasite line. In addition, two rif genes were found transcribed at early and late intra-erythrocyte stages independently of var gene transcription. Rif genes are organised in groups and inter-genomic conserved gene families, suggesting that RIFIN sub-groups may have different functional capacities. This conclusion is experimentally supported by group-specific rif transcription in parasites with different VSA and PfEMP1 expression phenotypes.