Few studies have examined the value of cytogenetic studies with flow cytometry (FC) in lymph node/extranodal tissue biopsies with suspected lymphoma. To evaluate this, G-banded karyotyping and/or fluorescence in situ hybridization (FISH) with FC immunophenotyping were performed on 185 lymph node or extranodal tissue biopsy specimens with suspected lymphoma. Complete cytogenetic analysis of lymph node/extranodal tissue was successful in 174 cases (94.1%) and 57.5% demonstrated chromosomal abnormalities. In 116 malignant lymphoma cases, 83.8% showed abnormalities. In 74 B cell lymphomas (B-NHL), abnormalities were more frequent in lymph node/extranodal tissues than in bone marrow by conventional cytogenetics (CC, 97.2 vs 26.1%), FISH (70.6 vs 17.6%), and FC (98.6 vs 28.4%). Three B-NHL diagnoses were confirmed by re-biopsy of lymph nodes due to the presence of abnormalities in the first biopsy, but no evidence of malignancy in pathological, FC, or IgH/TCR gene rearrangement analyses. In 29 T cell lymphomas (T-NHL), abnormalities were more frequent in lymph nodes than in bone marrow by CC (67.9 vs 21.4%) and FC (75.9 vs 27.6%) analyses. As expected, in 13 Hodgkin lymphoma cases, abnormalities were more frequent in lymph nodes than bone marrow by CC (41.7 vs 16.7%) and FC (30.8 vs 7.7%) analyses. In 56 reactive lymphoid hyperplasias (RLH), 7.1% had conventional clonal cytogenetic abnormalities. Two of these patients died of disease progression and two had their pathological diagnosis revised after the second review. These findings indicate that cytogenetic analysis combined with FC in lymph node/extranodal tissue biopsies can provide critical information in the auxiliary diagnosis of lymphoma.