Parageobacillus thermoglucosidasiusis a thermophilic Gram-positive bacterium, which is a promising host organism for sustainable bio-based production processes. However, to take full advantage of the potential ofP. thermoglucosidasius, more efficient tools for genetic engineering are required. The present study describes an improved shuttle vector, which speeds up recombination-based genomic modification by incorporating a thermostable sfGFP variant into the vector backbone. This additional selection marker allows for easier identification of recombinants, thereby removing the need for several culturing steps. The novel GFP-based shuttle is therefore capable of facilitating faster metabolic engineering ofP. thermoglucosidasiusthrough genomic deletion, integration, or exchange. To demonstrate the efficiency of the new system, the GFP-based vector was utilised for deletion of thespo0Agene inP. thermoglucosidasiusDSM2542. This gene is known to be a key regulator of sporulation inBacillus subtilis, and it was therefore hypothesised that the deletion ofspo0AinP. thermoglucosiadiuswould produce an analogous sporulation-inhibited phenotype. Subsequent analyses of cell morphology and culture heat resistance suggests that theP. thermoglucosidasius∆spo0Astrain is sporulation-deficient. This strain may be an excellent starting point for future cell factory engineering ofP. thermoglucosidasius, as the formation of endospores is normally not a desired trait in large-scale production.