Background: Detection of EGFR mutations in tumor tissue represents a standard testing procedure in patients with non-small cell lung cancer. Molecular testing of circulating tumor DNA (ctDNA) in plasma enables detection of mutations in cases where tumor specimens are unavailable or when monitoring of therapeutic responses is necessary. In addition, according to the recent literature, ctDNA better reflects the heterogeneity of the neoplastic cell population than isolated tumor lesion or metastasis. We report a national interlaboratory evaluation aimed at assessing the analytical quality of ctDNA EGFR testing in plasma across seven reference laboratories in the Czech Republic. Material and methods: Aliquots of 13 plasma samples were sent to 7 laboratories and consisted of commercially available 2 ml plasma specimens of genomic DNA with mutant allelic frequencies of 5, 0.5, 0.05, and 0% of the most common sensitizing mutations (deletion in exon 19, L858R) and the resistance mutation T790M. DNA extraction and EGFR testing were performed according to standard procedures. In 6/7 laboratories the cobas® EGFR Mutation Test v2 was used. One laboratory employed the Super-ARMS® EGFR Mutation Detection Kit. Results: In total, 91 genotypes were determined with an overall error rate of 24.2% (22/91). The overall error rates were 3.2% (2/63) for the 0.5% mutation frequency and 0% for the 5% mutation frequency (0/35), respectively. No false positive results were reported. The cobas® method achieved consistent results with the 0.05% mutation frequency for the exon 19 deletion. For L858R and T790M mutations, the threshold was above the 0.5% frequency. Conclusions: The results show that EGFR testing for ctDNA in plasma has limited sensitivity, especially for detection of the T790M mutation. Particularly, in ctDNA testing of very low mutated DNA plasma fractions (below 0.01%), emphasis should be placed on the use of highly sensitive molecular methods. The outcomes of this quality assessment confirm the need for rebiopsy in patients with negative plasma results because of a higher false negative rate in comparison to tissue testing.