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Background  The rodent malaria parasite  Plasmodium yoelii  is an important animal model for studying host-parasite interaction and molecular basis of malaria pathogenesis. Although a draft genome of  P. yoelii  yoelii  YM is available, and RNA sequencing (RNA-seq) data for several rodent malaria species (RMP) were reported recently, variations in coding regions and structure of mRNA transcript are likely present between different parasite strains or subspecies. Sequencing of cDNA libraries from additional parasite strains/subspecies will help improve the gene models and genome annotation.    Methods  Here two directional cDNA libraries from mixed blood stages of a subspecies of  P. yoelii  ( P. y. nigeriensis  NSM) with or without mefloquine (MQ) treatment were sequenced, and the sequence reads were compared to the genome and cDNA sequences of  P. y. yoelii  YM in public databases to investigate single nucleotide polymorphisms (SNPs) in coding regions, variations in intron–exon structure and differential splicing between  P. yoelii  subspecies, and variations in gene expression under MQ pressure.    Results  Approximately 56 million of 100 bp paired-end reads were obtained, providing an average of ~225-fold coverage for the coding regions. Comparison of the sequence reads to the YM genome revealed introns in 5′ and 3′ untranslated regions (UTRs), altered intron/exon boundaries, alternative splicing, overlapping sense-antisense reads, and potentially new transcripts. Interestingly, comparison of the NSM RNA-seq reads obtained here with those of YM discovered differentially spliced introns; e.g., spliced introns in one subspecies but not the other. Alignment of the NSM cDNA sequences to the YM genome sequence also identified ~84,000 SNPs between the two parasites.    Conclusion  The discoveries of UTR introns and differentially spliced introns between  P. yoelii  subspecies raise interesting questions on the potential role of these introns in regulating gene expression and evolution of malaria parasites.    Electronic supplementary material  The online version of this article (doi:10.1186/s12936-015-1081-9) contains supplementary material, which is available to authorized users.

UTR introns, antisense RNA and differentially spliced transcripts between Plasmodium yoelii subspecies