In this study, we investigated the pathogenicity, replication and tropism of the low pathogenic avian influenza (LPAI) strain A/chicken/Belgium/460/2019(H3N1) in adult SPF layers and young SPF males. The inoculated hens showed 58% mortality and a 100% drop in egg production in the second week post inoculation. The high viral loads in the cloacal samples coincided with the period of the positive immunohistochemistry of the oviduct, acute peritonitis and time of mortality, suggesting that the replication of H3N1 in the oviduct was a major component of the onset of clinical disease and increased level of excretion of the virus. In the inoculated young birds, the clinical signs were very mild with the exception of one bird. The results suggest that the time of replication of the virus was much shorter than in the adult layers; some of the young males did not show any proof of being infected at all. To conclude, the results of the study in young birds confirmed the intravenous pathogenicity test results but also showed that the clinical signs in adult layers were very severe. Based on the mortality without a bacterial component, complete drop of egg production and post mortem findings, this H3N1 strain is a moderately virulent strain, the highest category for LPAI strains. It is important to realize that if HPAI did not exist, this moderately virulent H3N1 virus would most likely to be considered as a very virulent virus.