Background <p>African swine fever (ASF), a devastating viral disease of pigs, is one of the main hindrances to pig farming in sub-Saharan Africa. Sporadic outbreaks of the disease occur annually in various regions of Uganda most of which remain undocumented. Confirmatory diagnosis of ASF is vital for instituting the disease prevention and control measures. Unfortunately, laboratory confirmatory diagnosis of ASF in Uganda is limited due to the high costs and expertise required. The aim of this study was to compare the level of agreement for positive detection of ASF virus by PCR and immunohistochemistry in archived formalin fixed, paraffin-embedded pig tissues from clinically suspected ASF cases in Uganda.</p> Results <p>More than a third of the archived pig lymphoid tissues, (lymph nodes 33.3%, 95/285 and spleen; 42.8%, 120/280) had lesions suggestive of ASF. On average, 17% and 15% of the archived samples had ASF viral DNA and antigen respectively. The positivity rate for ASF in the tested tissue samples was higher in spleen (PCR, 17.5% and IHC, 16.1%) than in other tissues. More than half (51.1%, 48/94) of the lymph nodes and 41.9% (49/117) of spleen tissues that had lesions suggestive of ASF tested positive for ASF using conventional PCR. There was a strong agreement (<i>kappa</i> = 0.93913 at 95% CI; 0.88634–0.99193) between IHC and PCR in diagnosis of ASF in formalin fixed paraffin embedded pig tissue samples examined.</p> Conclusion <p>ASF is still wide spread in Uganda and is one of the major threats to pig production in the country. PCR remain the most effective and reliable technique for diagnosing ASF in tissues even after a long period of archiving.</p>