Arachis paraguariensis Chodat and Hassl. is a wild peanut with very high morphogenic potential but limited rooting ability in vitro has hampered production of large number of surviving plantlets. The present study evaluated in vitro rooting and ex vitro survival of micro-shoots utilizing a 6×3×3×2 factorial experiment. Six A. paraguariensis genotypes were treated with three auxins at three concen- tration levels inside either 11.4 cm × 8.6 cm × 10.2 cm polyethylene terephthalate glycol (PETG) vessel or 2.5 cm × 15 cm glass tube. The auxin treatments included indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and -naphthaleneacetic acid (NAA) at 0.2, 0.6 and 1 mg L−1 . Across all auxin treatments, PETG ves- sel was superior to glass tube in terms of rooting percentage (68% vs 17%), and survival at acclimatization (38% vs 6%) respectively. Further analysis of plantlets cultured inside PETG vessel showed that rooting and post acclimatization survival were high on MS medium without any auxin (90% and 80%) and on 0.2 mg L−1 IBA (92% and 76%) or IAA (86% and 74%) respectively. Contrariwise, none of the shoots treated with 1 mg L−1 NAA survived acclimatization. The 0.2 mg L−1 of IAA and IBA treatments also exhibited high number of roots per plantlet (7 and 5), and lowest number of days to root initiation (11 and 12 days) respectively. Since rooting was delayed (26 days) on MS medium without auxin, we recommend supple- mentation of nutrient medium with 0.2 mg L−1 IBA or IAA whenever rapid rooting is desired. Overall, the results showed that genotype, auxin type, and auxin concentration significantly altered in vitro rooting of A. paraguariensis plantlets.