Isolation of spermatogonia from adult human testis is hampered by the limited selectivity of available markers. CD49f, CD90, and SSEA-4 were introduced as selection tools to isolate human spermatogonia (Conrad et al., 2008; Dym et al., 2009), whereas GFR-a1 and GPR-125 have been proved to be suitable for the prospective isolation of spermatogonial subsets (Grisanti et al., 2009; He et al., 2010, 2012). However, most of these markers, including CD49f and CD90, are not highly specific for spermatogonia (Izadyar et al., 2011) and the number of reports in which SSEA-4 is introduced as a selective target for spermatogonia isolation is limited (Izadyar et al., 2011). A drawback for the isolation and characterization of human spermatogonia is the limited availability of specimen of normal human testis. The isolation of spermatogonia is additionally hampered by the low frequency of these cells (∼0.02%–0.03%). In this study, we evaluated the suitability of combinations of known markers and novel targets for the prospective isolation of spermatogonia from adult human testis. Here we show that CD164 and SUSD2 can serve as attractive targets to prospectively isolate spermatogonia. In addition, we identified for the first time two distinct subsets of spermatogonia localized near the basement membrane, distinguished by their CD164 expression profile.