Structural and biophysical characterization of molecular mechanisms of disease-causing pathogens, such as Mycobacterium tuberculosis, often requires recombinant expression of large amounts highly pure protein. For the production of mycobacterial proteins, overexpression in the fast-growing and non-pathogenic species Mycobacterium smegmatis has several benefits over the standard Escherichia coli expression strains. However, unlike for E. coli, the range of expression vectors currently available is limited. Here we describe the development of the pMy vector series, a set of expression plasmids for recombinant production of single proteins and protein complexes in M. smegmatis. By incorporating an alternative selection marker, we show that these plasmids can also be used for co-expression studies. All vectors in the pMy vector series are available in the Addgene repository (www.addgene.com).