Feline immunodeficiency virus (FIV) is a widespread pathogen causing immunodeficiency in domestic cats and related wild cat species. The virus genome includes the main structural genes common to all retroviruses as well as accessory genes displaying essential functions during the viral life cycle. Expression of viral genes involves transcription of provirus genomes into full-length transcripts, which are partially processed into several spliced mRNA variants for the translation of particular proteins. Among several FIV isolates derived from domestic cats, notable differences in pathogenicity could be observed leading to identification of low and high pathogenic virus isolates. This study investigates the viral transcriptome of two differentially virulent FIV strains using second generation sequencing (SGS) technology. The expression levels of viral genes as detected by SGS were additionally determined by reverse transcription quantitative PCR analysis in order to compare two methods of mRNA quantification. The different properties of both methods, especially regarding normalization between samples, had to be considered when comparing the resulting data. SGS turned out to be a suitable technique for comparing mRNA transcription between both FIV infected cell lines and the identification of spliced viral transcripts. In contrast to this, the quantification of these spliced isoforms using SGS data was impeded by the short length of sequencing reads. In summary, SGS analysis revealed very consistent mRNA levels for the majority of viral genes between the low pathogenic Petaluma and the more highly pathogenic Glasgow 8 isolate. Notable differences among the two FIV strains could be observed in the viral mRNA splicing where Glasgow 8 displays similarities to the transcription pattern seen in the early stages of natural lentivirus infections. Thus, divergences in the regulation of post-transcriptional RNA processing might represent an additional contributor to the diverse pathogenic effects of individual FIV isolates. Taken together, this study aims to investigate the viral transcriptome as one part of the complex network of virus-host interactions, which will contribute to gaining deeper insights into FIV pathogenesis.