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Mucosa-associated lymphoid tissue (MALT) lymphoma mainly consists of three types of tumor B cells, small (centrocyte-like), scattered large transformed, and intraepithelial. However, it is difficult to differentiate tumor B cells from reactive B cells at the cellular level. We examined five cases of API2-MALT1 fusion-positive MALT lymphoma of the lung. A single paraffin section for each case was subjected to sequential retrieval of whole-slide imaging (WSI) data of hematoxylin and eosin (HE) staining, immunofluorescence staining for CD79a, and fluorescence in situ hybridization (FISH) for the MALT1 split. We counted the number of MALT1 split-positive or MALT1 split-negative cells among CD79a-positive cells. The MALT1 split was detected in 59, 46, and 76 % of small, large, and intraepithelial B cells, respectively. A review of the HE-WSI data showed that cytomorphological distinction between the MALT1 split-positive and MALT1 split-negative B cells was virtually impossible. None of CD79a-negative lymphoid cells, epithelial cells, and microvascular endothelial cells was positive for MALT1 splits. As API2-MALT1 fusion is an early and critical event in the lymphomatogenesis, our findings are best interpreted as that a considerable number of B cells, either small, large, or intraepithelial, are reactive cells and that it is difficult to distinguish cytomorphologically between tumor B cells and reactive B cells. These findings suggest that the tumor architecture may be the central factor for making a correct histopathological diagnosis of MALT lymphoma. The sequential WSI of HE staining, immunofluorescence staining, and FISH as described here is a useful tool for pathological analysis at the cellular level.