We formulate N -fold supersymmetry in quantum mechanical systems with reflection operators. As in the cases of other systems, they possess the two significant characters of N -fold supersymmetry, namely, almost isospectrality and weak quasi-solvability. We construct explicitly the most general 1-and 2-fold supersymmetric quantum mechanical systems with reflections. In the case of N = 2, we find that there are seven inequivalent such systems, three of which are characterized by three arbitrary functions having definite parity while the other four of which are by two. In addition, four of the seven inequivalent systems do not reduce to ordinary quantum systems without reflections. Furthermore, in certain particular cases, they are essentially equivalent to the most general two-by-two Hermitian matrix 2-fold supersymmetric quantum systems obtained previously by us.