This work aims to investigate the 64Cu isotope applicability for positron annihilation experiments in in situ mode. We determined appropriate characteristics of this isotope for defect studies and implemented them under aggressive conditions (i.e., elevated temperature, hydrogen environment) in situ to determine the sensitivity of this approach to thermal vacancies and hydrogen-induced defects investigation. Titanium samples were used as test materials. The source was obtained by the activation of copper foil in the thermal neutron flux of a research nuclear reactor. Main spectrometric characteristics (e.g., the total number of counts, fraction of good signals, peak-to-noise ratio) of this source, as well as line-shaped parameters of the Doppler broadening spectrum (DBS), were studied experimentally. These characteristics for 64Cu (in contrast to positron sources with longer half-life) were shown to vary strongly with time, owing to the rapidly changing activity. These changes are predictable and should be considered in the analysis of experimental data to reveal information about the defect structure. The investigation of samples with a controlled density of defects revealed the suitability of 64Cu positron source with an activity of 2–40 MBq for defects studies by DBS. However, greater isotope activity could also be applied. The results of testing this source at high temperatures and in hydrogen atmosphere showed its suitability to thermal vacancies and hydrogen-induced defects studies in situ. The greatest changes in the defect structure of titanium alloy during high-temperature hydrogen saturation occurred at the cooling stage, when the formation of hydrides began, and were associated with an increase in the dislocation density.