Next-generation sequencing (NGS) based on genomic DNA has been widely applied for gene rearrangement detection in patients with NSCLC. However, intergenic-breakpoint fusions, in which one or both genomic breakpoints localize to intergenic regions, confound kinase fusion detection. We evaluated the function of intergenic-breakpoint fusions with multiplex molecular testing approaches. NSCLCs with intergenic-breakpoint fusion identified by DNA-based NGS were analyzed by RNA-based NGS, immunohistochemistry (IHC), and fluorescence in situ hybridization. A total of 26 cases with single intergenic-breakpoint fusion were identified from a large cohort of NSCLCs using DNA-based NGS. Of the 26 cases, RNA-based NGS detected expressed fusion transcripts in 11 cases, but the genomic breakpoint position did not logically predict breakpoint of the fusion transcript in these cases, possibly owing to complex rearrangements (n = 5), alternative splicing (n = 2), and reciprocal rearrangement (n = 4). Nonetheless, no expressed fusion transcript was detected in five cases. Moreover, positive anaplastic lymphoma receptor tyrosine (ALK) finding was observed in three of the remaining 10 cases with IHC but not with RNA-based NGS. Three patients with intergenic-breakpoint ALK fusion with or without RNA-based NGS or IHC confirmation who received crizotinib treatment were found to have partial responses. However, one patient with intergenic-breakpoint ROS1, given the positive fluorescence in situ hybridization result, received crizotinib but developed progressive disease within 1 month, possibly owing to no functional fusion transcript detected by RNA-based NGS. Intergenic-breakpoint fusions detected by DNA sequencing confound kinase fusion detection in NSCLC, as functional fusion transcripts may be generated or not. Additional validation testing using RNA/protein assay should be performed in intergenic-breakpoint fusion cases to guide optimal treatment.