<p><strong>Background:</strong> Pre-invasive ocular surface squamous neoplasia (OSSN) are tumours of the ocular surface that include squamous neoplasms arising from the conjunctiva and the cornea, characterised by a multifactorial aetiopathogenesis. The role of human papillomavirus (HPV) in the aetiopathogenesis of OSSN is still controversial.</p><p><strong>Aim:</strong> This study aimed to determine the prevalence and genotypes of HPV in pre-invasive OSSN samples.</p><p><strong>Setting:</strong> The study setting was a tertiary laboratory in Northern Pretoria, South Africa, from 01 January 2012 to 31 December 2016.</p><p><strong>Methods:</strong> This was a retrospective cross-sectional study that utilised 58 formalin-fixed paraffin-embedded (FFPE) specimens of previously diagnosed pre-invasive OSSN cases. Immunohistochemistry (IHC) and molecular pathology techniques were used to detect HPV and data analysis was conducted using STATA statistical software.</p><p><strong>Results:</strong> The study sample had a higher proportion of females (60.3%; 35/58) compared to males (39.7%; 23/58) with an overall mean age of 41.8 ± 10.82 years. Human papillomavirus was detected in 20.7% (12/58) using IHC, 0% (0/58) by chromogenic in situ hybridisation, 19.0% (11/58) by linear array (LA) and 17.2% (10/58) by real-time polymerase chain reaction (RT-PCR). The HPV genotype commonly detected by LA and RT-PCR was HPV 16 at 12.1% and 8.6%, respectively.</p><p><strong>Conclusion:</strong> A minimal proportion of OSSN cases tested positive for HPV, predominantly exhibiting oncogenic high-risk HPV 16 positivity. The role of HPV in the oncogenesis of OSSN remains controversial.</p><p><strong>Contribution:</strong> This study contributes valued insights about HPV involvement in OSSN oncogenesis and HPV detection techniques in FFPE samples.</p>