Persistent infection with high-risk human papillomavirus (HPV) is recognized as the main cause for the development of anogenital cancers. This study prospectively evaluated the diagnostic performance of the novel Allplex-HPV28 assay with the Anyplex-II-HPV28 to detect and genotype HPV in 234 consecutive swabs and 32 biopsies of the anogenital tract from 265 patients with atypical findings in cytomorphological screening. Agreement in HPV-DNA detection between the Anyplex-II and Allplex-HPV28 assays was 99%. There was a notable diversity in the HPV-virome, with the most prevalent high-risk HPV types being 16, 53, 66, and 68. The agreement rates for detecting these genotypes exceeded 93% between the Anyplex-II and Allplex-HPV28 assays. Discrepancies in test results were solely noted for Anyplex-II-HPV28 results with a low signal intensity of "+", and for Allplex-HPV28 results with cycle thresholds of ≥36. The semi-quantitative analysis of HPV-DNA loads showed significant agreement between the Anyplex-II-HPV28 and Allplex-HPV28 assays (p < 0.001). Furthermore, HPV-DNA detection rates and mean HPV-DNA loads significantly correlated with the grade of abnormal changes identified in cytopathological assessment, being highest in cases of HSIL, condyloma accuminatum, and squamous cell carcinoma. Overall agreement rates for detecting specific HPV-types among the Anyplex-II and Allplex-HPV28 assays exceeded 99.5% in cases of atypical squamous cells, condyloma accuminatum, and squamous cell carcinoma. The novel Allplex-HPV28 assay shows good diagnostic performance in detecting and genotyping HPV commonly associated with anogenital cancers. Consequently, this assay could offer substantial potential for incorporation into future molecular screening programs for anogenital cancers in clinical settings.