Although disturbed cytokinesis of mononuclear Hodgkin (H) cells is thought to generate Reed‐Sternberg (RS) cells, differentiation of Hodgkin’s lymphoma (HL) cells is not fully understood. Recent studies indicate that cells found in a side population (SP) share characteristics of cancer stem cells. In this study we identified an SP in the HL cell lines, KMH2 and L428. This SP almost entirely consists of distinct small mononuclear cells, whereas the non‐SP is a mixture of relatively large cells with H or RS cell‐like morphology. Culture of the small mononuclear cells in the SP from KMH2 generated a non‐SP. Single cell culture of the SP cells generated large cells with H or RS cell‐like morphology. We found that CD30 overexpression and constitutive nuclear factor‐κB (NF‐κB) activity, both of which are characteristics of HL cells, are shared between the SP and non‐SP cells for both KMH2 and L428. Inhibition of NF‐κB induced apoptosis in both fractions, whereas the SP cells were resistant to a conventional chemotherapeutic agent doxorubicin. The results show that HL cell lines contain an SP, that is enriched for distinct small mononuclear cells and generates larger cells with H and RS cell‐like morphology. The results also stress the significance of NF‐κB inhibition for eradication of HL cells. (Cancer Sci 2010; 101: 2490–2496)