Background: High-risk Human Papillomavirus (HPV) testing is replacing cytology in cervical cancer screening as it is more sensitive for preinvasive cervical lesions. However, the bottleneck of HPV testing is the many false positive test results (positive tests without cervical lesions). Here, we evaluated to what extent these can be explained by cross-reactivity, i.e. positive test results without evidence of high-risk HPV genotypes. The patterns of cross-reactivity have been thoroughly studied for hybrid capture II (HC2) but not yet for newer HPV assays although the manufacturers claimed no or limited frequency of cross-reactivity. In this independent study we evaluated the frequency of cross-reactivity for HC2, cobas, and APTIMA assays. Methods: Consecutive routine cervical screening samples from 5022 Danish women, including 2859 from women attending primary screening, were tested with the three evaluated DNA and mRNA HPV assays. Genotyping was undertaken using CLART HPV2 assay, individually detecting 35 genotypes. The presence or absence of cervical lesions was determined with histological examinations; women with abnormal cytology were managed as per routine recommendations; those with normal cytology and positive high-risk HPV test results were invited for repeated testing in 18months. Results: Cross-reactivity to low-risk genotypes was detected in 109 (2.2%) out of 5022 samples on HC2, 62 (1.2%) on cobas, and 35 (0.7%) on APTIMA with only 10 of the samples cross-reacting on all 3 assays. None of the 35 genotypes was detected in 49 (1.0%), 162 (3.2%), and 56 (1.1%) samples, respectively. In primary screening at age 30 to 65years (n=2859), samples of 72 (25%) out of 289 with high-risk infections on HC2 and<CIN2 histology were due to cross-reactivity. On cobas, this was 106 (26%) out of 415, and on APTIMA 48 (21%) out of 224. Conclusions: Despite manufacturer claims, all three assays showed cross-reactivity. In primary cervical screening at age ≥30years, cross-reactivity accounted for about one quarter of false positive test results regardless of the assay. Cross-reactivity should be addressed in EU tenders, as this primarily technical shortcoming imposes additional costs on the screening programmes.