Simple SummaryThe morphological diagnosis of peripheral lung lesions is challenging due to the anatomical location of these lesions and to the biopsy techniques that are currently available. Transbronchial forceps biopsies guided by fluoroscopy alone, or additional with radial mini probe endobronchial ultrasound (RP-EBUS), were well-known standardised procedures with an unsatisfactory diagnostic yield. Transbronchial cryobiopsy is a novel technique of lung parenchyma biopsy for the diagnosis of interstitial lung diseases, which has a very high diagnostic yield (80–96%) and relatively low incidence of complications (3–26%). Due to these reasons, transbronchial cryobiopsy guided by RP-EBUS and fluoroscopy in peripheral lung lesions are under active investigation, but still, there is limited data as only a few clinical trials of transbronchial cryobiopsy are published around the world. Our study showed that transbronchial cryobiopsy is a potentially safe procedure when obtaining a larger size of samples. Furthermore, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were 85.1%, 100%, 100%, 12.5%, and 93% for TBCB as well as 91.5%, 100%, 100%, 20.0% and 96.7% for the combined procedures, respectively.Transbronchial cryobiopsy (TBCB) is being studied in the diagnosis of peripheral lung lesions; however, there are only a few clinical studies around the world. The aim of our study was to evaluate the diagnostic values and safety of transbronchial cryobiopsy for radiologically suspected peripheral lung cancer. The prospective clinical study was executed from September 2019 to September 2021 at a tertiary clinical centre in Lithuania. A total of 48 patients out of 102 underwent combined procedures of transbronchial forceps biopsy (TBFB) and TBCB. Diagnostic values and safety outcomes of TBFB and TBCB were analysed. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were 72.9%, 100%, 100%, 7.7%, and 88.0% for TBFB, 85.1%, 100%, 100%, 12.5%, and 93% for TBCB, as well as 91.5%, 100%, 100%, 20.0% and 96.7% for the combined procedures, respectively, with a significantly higher accuracy for cryobiopsies compared to forceps biopsies (p < 0.05). The diagnostic values for transbronchial cryobiopsies were similar, irrespective of the radial mini probe endobronchial ultrasound (RP-EBUS) position, lesion size or bronchus sign, however, the sensitivity of the combined procedures in cases with RP-EBUS adjacent to the target was significantly higher compared to TBFB (86.2% vs. 64.3%, p = 0.016). Samples of cryobiopsies were significantly larger than forceps biopsies (34.62 mm2 vs. 4.4 mm2, p = 0.001). The cumulative diagnostic yield of transbronchial cryobiopsy was 80.0% after the second biopsy and reached a plateau of 84.1% after four biopsies. No severe bleeding, pneumothorax, respiratory failure or death was registered in our study. TBCB is a potentially safe procedure, which increases diagnostic values in diagnosing peripheral lung lesions compared to TBFB.