Whilst molecular methods which detect viral nucleic acid have replaced more traditional assays such as virus isolation (VI) as frontline diagnostic assays for avian influenza virus (AIV), the ability to isolate live viruses from samples remains critical for all downstream research and virus categorisation activities. VI, when successful, enables detailed functional characterisation of the AIV strain and provides essential reference material for diagnostic testing for animal and public health. However, VI is resource intensive, requiring specialist facilities, reagents, trained staff and time. Therefore, understanding the factors which contribute to successful VI is paramount in targeting efforts. In this study, using a range of clinical samples collected from wild birds during the high pathogenicity AIV 2020-2022 panzootic, we assessed factors which limited successful VI including sample collection time, sample type, subtype, originator species and sample viral RNA level. Although virus stability is highly temperature-sensitive and cold chain failure or prolonged exposure of carcasses or samples to ambient temperatures could negatively impact virus viability despite strong RT-PCR Cq values, we propose an upper RT-PCR Cq value, which increases the likelihood for optimal VI when handling poor quality material. Understanding these factors enables efficient triage for VI to direct resources for maximum success.