Murine gammaherpesvirus 68 (MHV68) establishes latency mainly in B cells and causes lymphomas reminiscent of human gammaherpesvirus diseases in laboratory mice. To study the molecular mechanism of virus infection and how the viral determinants control cell and eventually cause tumorigenesis, readily available latently infected cell lines are essential. For in vitro MHV68 latency studies, only two cell culture systems have been available. Gammaherpesviruses are known to infect developing B cells and macrophages, therefore we aimed to expand the MHV68 latently infected cell line repertoire. Here, several latently infected immature B cell and macrophage-like cell line clones were generated. Hygromycin-resistant recombinant MHV68 was isolated from a laboratory-made latent cell line, HE2.1, and propagated to develop stable cell lines that carry the viral genome under hygromycin selection. Subclones of these cells lines were analyzed for viral miRNA expression by TaqMan qPCR and assessed for expression of a lytic viral transcript M3. The cell lines maintain the viral genome as an episome shown by the digestion-circularization PCR assay. Latently infected cell lines generated here do not express viral miRNAs higher than the parental cell line. However, these cell lines may provide an alternative tool to study latency mechanisms and miRNA target identification studies.