A semiconductor device may include a substrate having a channel recess therein, a plurality of spaced apart shallow trench isolation (STI) regions in the substrate, and source and drain regions spaced apart in the substrate and between a pair of the STI regions. A superlattice channel may be in the channel recess of the substrate and extend between the source and drain regions, with the superlattice channel including a plurality of stacked group of layers, and each group of layers of the superlattice channel including stacked base semiconductor monolayers defining a base semiconductor portion and at least one non-semiconductor monolayer constrained within a crystal lattice of adjacent base semiconductor portions. A replacement gate may be over the superlattice channel.