Tumor spread through air spaces (STAS) has three morphologic subtypes: single cells, micropapillary clusters, and solid nests. However, whether their respective clinical significance is similar remains unclear. We retrospectively reviewed 803 patients with resected non-small cell lung cancer (NSCLC) from January to December 2009. Recurrence-free survival (RFS) and overall survival (OS) were compared among patients stratified by STAS subtypes. We also performed a prospective study of NSCLC resection specimens to evaluate the influence of a prosecting knife on the presence of STAS subtypes during specimen handling (83 cases). STAS was found in 370 NSCLCs (46%), including 47 single cell STAS (13%), 187 micropapillary cluster STAS (50%), and 136 solid nest STAS (37%). STAS-negative patients had significantly better survival than patients with micropapillary cluster STAS (RFS: < 0.001; OS: < 0.001) and solid nest STAS (RFS: < 0.001; OS: < 0.001), but similar survival compared with those with single cell STAS (RFS: = 0.995; OS: = 0.71). Multivariate analysis revealed micropapillary cluster (RFS: < 0.001; OS: < 0.001) and solid nest STAS (RFS: = 0.001; OS: = 0.003) to be an independent prognostic indicator, but not for single cell STAS (RFS: = 0.989; OS: = 0.68). Similar results were obtained in subgroup analysis of patients with adenocarcinoma. The prospective study of NSCLC specimens suggested that 18 cases were considered as STAS false-positive, and most were singe cell pattern (13/18, 72%). Single cell STAS was the common morphologic type of artifacts produced by a prosecting knife. A precise protocol of surgical specimen handling is required to minimize artifacts as much as possible.