High transformation and gene editing efficiencies in sorghum-produced, transgene-free SDN1-edited plants exhibit precise mutations, reduced germination stimulants, and enhanced resistance to Striga infection. Sorghum (Sorghum bicolor L.) is a primary food staple grain for millions in Sub-Saharan Africa (SSA). It is mainly constrained by the parasitic weed Striga, which causes up to 100% yield losses and affects over 60% of cultivable farmlands and livelihoods. In this study, CRISPR/Cas9 technology is utilized to induce mutations in core strigolactone (SL) biosynthetic genes, i.e., CCD7, CCD8, MAX1, in addition to an uncharacterized gene (DUF) in the fine-mapped 400 kb lgs1 region in sorghum to develop durable Striga resistance. Two sorghum cultivars were delivered with the expression cassettes through immature embryo-based Agrobacterium-mediated transformation. Our study demonstrated transformation and gene editing efficiencies of ~ 70 and up to 17.5% (calculated based on the numuber of established plants), respectively, in two sorghum genotypes. Subsequent analysis of homozygous E0 lines in the E1 generation confirmed stable integration of mutations for all targeted genes. Loss-of-function mutations in the CCD7, CCD8, MAX1, and DUF genes led to a significant downregulation of the expression of associated genes in the SL biosynthetic pathway. The phenotypic analysis of edited lines revealed changes in phenotypic patterns compared to wild-type plants. Analysis of root exudates showed significant reductions in SL production in edited lines compared to wild-type plants. Striga infection experiments demonstrated delayed or reduced emergence rates of Striga in edited lines with lower SL production, highlighting the potential for genetically altering SL production to control Striga infestations. This study provides insights into the functional roles of CCD7, CCD8, MAX1, and DUF genes in sorghum towards reduced and/or altered SL production and improved resistance to Striga infestations.