Next-generation sequencing (NGS) technology is routinely employed to detect clinically significant variants in the field of precision medicine. Formalin-fixed paraffin-embedded (FFPE) tissues remain a widely used source of genetic material for diagnostic purposes due to their long-term storage stability, which preserves the architecture of tumour tissues. However, the degradation of nucleic acids (NAs) that occurs during the fixation process can lead to unreliable results or hinder analysis. Considering the challenges posed by the quality and quantity of nucleic acids extracted from FFPE samples, the present study aimed to compare paired fresh-frozen (FF) and FFPE samples. The present study also aimed to assess the concordance rate of quality control metrics of sequenced libraries and identify variants and biomarkers between FFPE and FF tissues. The Illumina® TruSight Oncology 500 assay (TSO 500; Illumina, Inc.) was used to conduct comprehensive genomic profiling of samples from patients with various cancer types. All identified alterations were annotated using the Pierian Dx Clinical Genomics Workspace version 6.20 (PierianDx CGW). A total of 138 DNA and 138 RNA analyses on 69 paired samples were performed. These results demonstrated the significant potential of FF tissue as a primary source of higher-quality genetic material to detect small variants, microsatellite instabilities and the tumour mutational burden, using the TSO 500 assay compared with FFPE samples. This approach could effectively reduce the issues associated with poor-quality NAs extracted from FFPE samples. Based on our findings of lower concordance in the detection of splice variants, fusions and copy number variants in paired samples, we recommend that future studies using the TSO 500 assay should focus directly on this issue.