In this study we have used high-throughput transcriptomic sequencing of the Finnish Large White breed to identify gene polymorphisms and expression patterns related to reproduction. The dataset contains sequences from samples collected from immotile short-tail sperm defect (ISTS)-affected individuals and control animals. ISTS-affected boars are infertile due to immotile and short sperm tails (Andersson et al. 2000; Sukura et al. 2002). The ISTS phenotype is caused by an altered splicing pattern of exon 30 of the SPEF2 gene, which results in premature translation stop codons (Sironen et al. 2006). The cause for the altered splicing pattern was shown to be a full-length L1 insertion within intron 30 (Sironen et al. 2006, 2007). We have investigated and identified an association between the L1 insertion and litter size in the Finnish Large White pig population (Sironen et al. 2012), which may be caused by a significant decrease in PRLR expression in the ISTS-affected and carrier sows (Sironen et al. 2014). Because the mechanisms underlying the causative mutation are known, we assume that the ISTS mutation does not induce changes in the expression of other genes between the ISTS-affected and control animals. The present investigation tested the hypothesis that RNAseq data from two testis and two oviduct samples can be used, first for the identification of highly expressed genes in these tissues, second for discerning genes specifically affecting male or female reproduction, and third for characterizing gene polymorphisms within identified genes. These genetic polymorphisms serve as candidate variants in association analysis and consequently potential biomarkers for pig fertility. Furthermore, we have used the data for the discovery of 55 previously unannotated genes using a novel in-house analysis pipeline.