Natural microbial products are important resources for pharmaceuticals. Geldanamycin derivatives, as heat shock protein 90 (Hsp90) inhibitors, hold promise in cancer therapy. We developed a LC-MS-based dereplication method to identify geldanamycin derivatives from actinomycetes culture broths. Using a LTQ XL ion trap mass spectrometer, we investigated fragmentation patterns of geldanamycins under electrospray ionization (ESI) LC-MS, generated in silico fragmentation libraries with Mass Frontier software, and analyzed ~500 crude extracts from in-house-collected actinomycetes. A Streptomyces sp. W1348 strain was identified, producing five decarbamoyl 4,5-dihydrogeldanamycin derivatives (1–5). Compounds 1–3 were known, while 4 and 5 were new (compound 4 was unstable and spontaneously converted to 1 during purification). NMR analysis confirmed their decarbamoyl (C-7 free hydroxyl) and 4,5-dihydro (hydrogenated C-4/C-5 double bond) features, suggesting the strain lacks a functional carbamoyltransferase gene. New compound 5 (7-O-descarbamoyl-17-O-demethylreblastatin) provided insight into geldanamycin biosynthesis, revealing that C-17 hydroxylation precedes C-18/C-21 oxidation.