Enumeration and isolation of Streptococcus salivarius subsp. thermophilus from cheese is challenging, due to the relatively high number of species it may host. We describe medium SPY9.3 for the cultivation of S. salivarius subsp. thermophilus from cheese. The medium and related incubation conditions (SPY) was compared with 2 other protocols, M17 and ST: sensitivity was assessed by parallel cultivation of 55 strains of S. salivarius subsp. thermophilus, and selectivity by (i) parallel cultivation of 60 strains belonging to 20 different non-target species and sub-species and (ii) isolating bacteria from 3 raw-milk cheeses. Colony counts were similar on SPY9.3 and M17 (mean difference 0.07 log(cfu/mL), p > 0.001) and significantly higher on ST than on M17 and SPY9.3 (mean differences 0.42 and 0.48 log(cfu/mL), respectively, p < 0.001). SPY was more specific than ST and M17, with respectively 20%, 40%, and 50% of the investigated non-target species able to grow. S. salivarius subsp. thermophilus, Enterococcus spp., and Staphylococcus aureus were indistinguishable using all 3 protocols. Only SPY avoided growth of Lactobacillus delbrueckii subsp. lactis. Finally, ST and SPY displayed higher recoveries of S. salivarius subsp. thermophilus colonies from cheese than M17 (5.6, 5.5, and 3.0 adjusted log(cfu/mL), respectively) and the lowest proportion of non-specific isolates. The protocol described here and based on SPY9.3 presents a promising alternative to existing protocols for the enumeration and isolation of S salivarius subsp. thermophilus from cheese or other complex fermented products. Copyright © 2020 Agroscope. Published by Elsevier Ltd.. All rights reserved.