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Deformation twinning is a phenomenon that causes local shear strain concentrations, with the material either experiencing elongation (and thus a tensile stress) or contraction (compressive stress) along the stress directions. Thus, in order to gauge the performance of the alloy better, it is imperative to predict the activation of twinning systems successfully. The present study investigates the effects of deformation by cold-rolling on the {332}<113> and {112}<111> twin variant activation in a Ti-30Nb-12Zr-5Ta-2Sn-1.25Fe (wt.%) (TNZTSF) alloy. The Ti-30Nb-12Zr-5Ta-2Sn-1.25Fe (wt.%) alloy was synthesized in a cold crucible induction levitation furnace, under an argon-controlled atmosphere, using high-purity elemental components. The TNZTSF alloy was cold-deformed by rolling, in one single step, with a total deformation degree (thickness reduction) of ε ≈ 1% (CR 1), ε ≈ 3% (CR 3), and ε ≈ 15% (CR 15). The microstructural investigations were carried out with the SEM-EBSD technique in order to determine the grain morphology, grain-size distribution, crystallographic orientation, accumulated strain-stress fields and Schmid Factor (SF) analysis, all necessary to identify the active twin variants. The EBSD data were processed using an MTEX Toolbox ver. 5.7.0 software package. The results indicated that the TNZTSF alloy's initial microstructure consists of a homogeneous β-Ti single phase that exhibits equiaxed polyhedral grains and an average grain-size close to 71 μm. It was shown that even starting with a 1% total deformation degree, the microstructure shows the presence of the {332}<113> twinning (( 233 )[ 3 ¯ 11 ] active twin variant; Schmit factor SF = −0.487); at a 3% total deformation degree, one can notice the presence of primary and secondary twin variants within the same grain belonging to the same {332}<113> twinning system (( 32 3 ¯ )[ 1 3 ¯ 1 ¯ ] primary twin variant—SF = −0.460; ( 23 3 ¯ )[ 3 ¯ 1 1 ¯ ] secondary twin variant—SF = −0.451), while, at a 15% total deformation degree, besides the {332}<113> twinning system, one can notice the activation of the {112}<111> twinning system (( 1 1 ¯ 2 )[ 1 ¯ 11 ] active twin variant—SF = −0.440). This study shows the {332}<113> and {112}<111> twinning variant activation during cold-deformation by rolling in the case of a Ti-30Nb-12Zr-5Ta-2Sn-1.25Fe (wt.%) (TNZTSF) alloy. [ABSTRACT FROM AUTHOR]