Trancriptome analysis is commonly employed to evaluate biological processes in both human and livestock species. One of the prerequisites for this type of analysis is the possession of high purity, intact RNA. Postmortem tissue collection has inherent time delays and hence it is important to understand the temporal variation, both in the stability of total RNA and individual gene transcripts, with respect to particular tissues. The objective of this experiment was to both qualitatively and quantitatively assess the integrity of both total and mRNA species derived from ovine liver, spleen, thyroid, skeletal muscle, ileum and perirenal adipose tissue, which has been stored at ambient temperature and sampled at time points 0, 3, 6 and 9 h post-mortem. One hour after parturition, 6 lambs (5.12 ± 0.27 kg) were euthanized. Samples were collected from the liver, spleen, thyroid, skeletal muscle, ileum and perirenal adipose tissue and stored on a sterile Petri dish at ambient temperature. Approximately 1–2 g of tissue was then harvested at each time point (0, 3, 6, 9h), held for 24h in RNAlater and then stored at −80°C. The quality and quantity of total RNA was assessed on the NanoDrop spectrophotometer and Agilent 2100 Bioanalyzer, respectively. While postmortem sampling time had no effect on RNA quantity (P ≥ 0.05) in 5 of the 6 tissue types analyzed, the RNA integrity number decreased over time and was significantly lower at 6 and/or 9 h postmortem in the spleen, thyroid, skeletal muscle, ileum and perirenal adipose tissues relative to the 0-h time point (P < 0.05). A reduction in the normalization factor in the liver, spleen, ileum and perirenal adipose tissues was observed over the time period (P < 0.05). In summary, the stability of total RNA remained intact within the first 3 h postmortem, regardless of tissue type, however tissue specific variation was evident in the RNA integrity across the 4 postmortem sampling times.