Defect characterization of as-grown Zn1−xBexSe compound semiconductors was studied by positron lifetime and photoluminescence measurements. We obtain both experimental and theoretical evidence that the bulk lifetime of free positrons decreases linearly with Be alloying. The average positron lifetime increases with the temperature, indicating that both vacancies and negative ions trap positrons. The decomposition of the lifetime spectra shows that the positron lifetime of the vacancy decreases with an increase in Be content, as predicted by theoretical calculations. The concentration of vacancies and negative ions obtained from positron measurements follows the same trend with Be alloying. A comparison of positron measurements with theoretical calculations and photoluminescence experiments in Zn vapor annealed samples indicates that the vacancy corresponds to a Be vacancy. Besides, the negative ions trapping positrons at low temperatures have been attributed to Be interstitial atoms. The intensity of the B photoluminescence band correlates with the concentration of Be vacancies and Be interstitial atoms. We thus conclude that the electron levels of those defects are involved in the optical transition that leads to the B photoluminescence band.