The establishment of clinically relevant human myeloma cell lines is central for our understanding of myeloma pathogenesis and development of novel therapies for the disease. Unfortunately, most available lines were generated from extramedullary sites, harbored multiple genetic abnormalities and categorized as non-hyperdiploid. In contrast, hyperdiploid myeloma cell lines, which represent more than 50% of patients, are rare. We established procedures for establishment of stroma-dependent myeloma lines by passaging primary myeloma cells, in severe combined immunodeficient-human (SCID-hu) or SCID-rab mice followed by maintenance in co-culture with stromal cells. We described the establishment and characterization of two hyperdiploid (LD and CF) and two non-hyperdiploid (JB and BN) cell lines. Using our animal models, we also established bortezomib-sensitive and -resistant BN lines. These cell lines were cellularly, phenotypically and molecularly characterized using flow cytometry immunophenotyping, DNA content, G-band and multicolor spectral karyotyping (SKY) and global gene expression profiling. All four cell lines were infected with lentiviral-expressing luciferase for detection of tumour cells at high sensitivity level and for monitoring myeloma growth in co-cultures and in vivo by live animal imaging. These myeloma cell lines and the procedures used for their establishment provide essential tools for studying myeloma biology and therapy.