Single molecular localization microscopy (SMLM) is a useful tool in biological observation with sub-10-nm resolution. However, SMLM is incapable of discerning two molecules within the diffraction-limited region unless with the help of a stochastic on–off switching scheme which yet entails time-consuming processes. Here, we produce a novel kind of focal spot pattern, called sub-diffraction dark spot (SDS), to localize molecules within the sub-diffraction region of interest. In our proposed technique nominated as sub-diffracted dark spot localization microscopy (SDLM), multiple molecules within the diffraction-limited region could be distinguished without the requirement of stochastic fluorescent switches. We have numerically investigated some related impacts of SDLM, such as detection circle diameter, collected photon number, background noise, and SDS size. Simulative localization framework has been implemented on randomly distributed and specifically structured samples. In either two- or three-dimensional case, SDLM is evidenced to have ∼2nm localization accuracy.