Hydrogels are an especially appealing class of biomaterials for gene delivery vehicles as they can be introduced into the body with minimally invasive procedures and are often applied in tissue engineering and regenerative medicine strategies. In this study, we show for the first time the use of an injectable alginate hydrogel for controlled delivery of lentivectors in the skeletal muscle of murine hindlimb. We propose to alter the release rates of lentivectors through manipulation of the molecular weight distribution of alginate hydrogels. The release of lentivector was tested using two different ratios of low and high molecular weight (MW) alginate polymers (75/25 and 25/75 low/high MW). The interdependency of lentivector release rate and alginate degradation rate was assessed in vitro. Lentivector-loaded hydrogels maintained transduction potential for up to one week in vitro as demonstrated by the continual transduction of HEK-293T cells. Injection of lentivector-loaded hydrogel in vivo led to a sustained level of transgene expression for more than two months while minimizing the copies of lentivirus genome inserted into the genome of murine skeletal muscle cells. This strategy of spatiotemporal control of lentivector delivery from alginate hydrogels may provide a versatile tool to combine gene therapy and biomaterials for applications in regenerative medicine.