Self-destructive cooperators, which sacrifice themselves for others, challenge traditional group selection theory, as costs often exceed individual benefits. We predict self-destructive cooperators can persist in highly segregated environments where populations are primarily divided into homogenous groups originating from one or two founders. In such contexts, the benefits of self-destructive cooperators remain within homogeneous groups of self-destructive cooperators, preserving the sacrifice value and ensuring its maintenance. To validate our hypothesis, we employ a synthetic self-destructive cooperators-cheaters system and develop automated experiments to monitor and operate the subgroups with diverse growth behaviors due to strong segregation. Ultimately, we demonstrate self-destructive cooperators is maintained under strong segregation. High stress further enhances self-destructive cooperators by reducing the benefits received by cheaters in heterogeneous subgroups. This study advances group selection theory and automation in evolutionary research.