Pepper (Capsicum annuum L.) is a popular vegetable and condiment consumed around the world. In the Guizhou Province of China, peppers are the most commonly grown crop on 300,000 planted hectares. A variety of diseases routinely occur on peppers in this province, resulting in yield losses (Liu et al., 2022). Root rot is one of the most common symptoms and produces poor root growth and wilting of pepper. In April 2023, symptomatic pepper plants displaying stunting, dwarfism, wilting, and root browning were collected from five fields in Guizhou, with disease incidence ranging from 10% to 20%. The collected rotten roots were cleaned with sterilize distilled water and placed in selective V8 juice agar (V8A) medium (15% clarified V8 juice with 2.5 g/L CaCO3 and 2% agar) containing nystatin, ampicillin, rifampicin, and miconazole, and incubated at 25℃ for 1 to 2 days (Morita and Tojo, 2007). Eight isolates with similar colony morphology were transferred to V8A medium via hyphal tipping, and incubated at 25℃ in the dark. Colony and sexual structures were observed using a microscope. Mycelium was aseptate and formed white cottony colonies. Globose, intercalary, or terminal hyphal swellings were observed with a diameter of 20.5 to 25 µm (average: 22 µm), and aplerotic oospores had a diameter of 15 to 20 µm (average: 17.5 µm) with a wall thickness of approximately 2 µm. Three representative isolates HSLJ-3, LJG-1, and LJY-2 were chosen for further molecular identification. Sequences of the internal transcribed spacer (ITS) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes were identified using primer sets ITS4/ITS5 (White et al., 1990) and OomCoxI-Levup/OomCoxI-Levlo (Robideau et al., 2011), respectively. All sequences were deposited in GenBank (accession nos. OR554005, PP083310, and PP083420 for ITS, and OR529247, PP093821 and PP093822 for cox1). BLAST analysis revealed all ITS and cox1 sequences exhibited 100% identity with Globisporangium ultimum (Pythium ultimum) isolate BR850 (GenBank accession nos. HQ643892.1 and HQ708933.1 for ITS and cox1, respectively). Phylogenetic analysis was performed by the maximum-likelihood method on the CIPRES web portal (https://www.phylo.org/portal2/login!input.action, accessed on 9 January 2024). For pathogenicity tests, each isolate was cultured in V8A medium containing 50 autoclaved wheat seeds at 25℃ for 7 days. Budding pepper seedling (cv. Huaxi) was transplanted into a 0.4 L pot containing sterilized commercial potting mix (Seedling Cultivation Substrate, Hunan Xianghui Agricultural E-commerce Co., Ltd.) which was saturated with deionized water. Eight infected and non-infected wheat seeds were placed near the roots of five pepper seedlings, respectively. Plants were placed in an artificial climate chamber, with a 14 h photoperiod and approximately 75% relative humidity at 25℃. After 14 days, inoculated seedlings showed symptoms of stunting, wilting, and rotting roots similar to those observed in the field. No disease was observed on the non-inoculated control plants. The pathogen was isolated from infected pepper roots and confirmed as G. ultimum by morphological and molecular analyses as previously described. This is the first report of G. ultimum causing root rot on pepper in Guizhou, China. This finding is critical to the discover of treatment options for this pathogen, thereby improving management practices to reduce yield losses in pepper.