PD-L1 expression assessed by immunohistochemical staining is used for the selection of immunotherapy in non-small cell lung cancer (NSCLC). Appropriate validation of PD-L1 expression in cytology specimens is important as cytology is often the only diagnostic material in NSCLC. In a previous study comprising two different cohorts of paired biopsies and cytological specimens, we found a fairly good cyto-histological correlation of PD-L1 expression in one, whereas only a moderate correlation was found in the other cohort. Therefore, that cohort with additional new cases was now further investigated for the impact of preanalytical factors on PD-L1 concordance in paired biopsies and cytological specimens. A total of 100 formalin-fixed paraffin-embedded cell blocks from 19 pleural effusions (PE), 17 bronchial brushes (BB), and 64 bronchoalveolar lavage (BAL) and concurrent matched biopsies from 80 bronchial biopsies and 20 transthoracic core biopsies from NSCLC patients were stained using the PD-L1 28-8 assay. Using the cutoffs ≥1%, ≥5%, ≥10%, and ≥50% positive tumour cells, the overall agreement between histology and cytology was 77-85% (κ 0.51-0.70) depending on the applied cutoff value. The concordance was better for BALs (κ 0.53-0.81) and BBs (κ 0.55-0.85) than for PEs (κ -0.16-0.48), while no difference was seen for different types of biopsies or histological tumour type. A high number of tumour cells (>500) in biopsies was associated with better concordance at the ≥50% cutoff. In conclusion, the study results suggest that PEs may be less suitable for evaluation of PD-L1 due to limited cyto-histological concordance, while a high amount of tumour cells in biopsies may be favourable when regarding cyto-histological PD-L1 concordance.