The aim of this study is to investigate the effect of salep species grown in different regions of Turkey on the rheological and microstructural properties of ice cream mix and the thermal and sensory properties of ice cream. For this purpose, ten different types of salep grown in different regions (Mersin, Yozgat, Muğla, Kahramanmaraş, Adana, Van, Muş) in the natural microflora of Turkey were used in a formulation of an ice cream mix. The flow behavior, frequency sweep, and 3-ITT rheological properties of ice cream mixes were studied. All samples showed shear thinning, viscoelastic solid-like, and recoverable character. The K and n values for the ice cream mixes were determined as 0.03-35.08 Pas n and 0.33-0.80, respectively, and significantly differed according to salep varieties (p < 0.05). The zeta potential values of ice cream mix samples ranged from -25.87 mV to -33.95 mV and were significantly affected by salep varieties (p < 0.05). The use of different salep varieties significantly affected the thermal properties of ice cream such as freezing point temperature (Tf), temperature range (ΔT), and enthalpy of fusion (ΔH f ). In conclusion, the results of this study showed that the use of different salep varieties can significantly affect the rheological, thermal, and sensory properties of ice cream, and the selection of salep varieties may be vital for the desired quality of ice cream.