Maize (Zea mays L.) with symptoms of premature leaf senescence and wilting was observed in two commercial fields, in Santarem (Portugal) and Almodovar del Rio (Spain), in the summer of 2014. Similar symptoms including brown lesions of lower internodes and disintegrated stalk pith tissues occurred again in two commercial fields in Almacelles and Biota (Spain) in the summers of 2016 and 2017, respectively. Depending on the field, estimated disease incidence varied between 10 and 30%. Lodging was observed on 10 to 20% of maize plants in the two commercial fields following rain and strong wind. Four symptomatic plants were collected from each of the four fields. Their first internodes were excised, surface sterilized for 5 min in 10% sodium hypochlorite solution, rinsed in water, and air dried. Cross sections were incubated in potato dextrose agar (PDA) at 25°C in darkness. Fungal colonies with similar morphological features were isolated from all the samples. One colony from each location was selected and two single-spore isolates obtained. Colonies grown on PDA were white-beige, flat with floury appearance at the top, and light gray turning to irregular shapes of black with age at the bottom. Globose pycnidia similar to those of Stenocarpella maydis (de la Riva et al. 2019) were observed. The fungus was tentatively identified as Phaeocytostroma ambiguum (Mont.) Petr. in Petr. & Syd. (syn. Phaeocytosporella zeae G. L. Stout) (White 1999). The region consisting of the 5.8S ribosomal DNA and internal transcribed spacers 1 and 2 was amplified with the primer set ITS5/ITS4. PCR products were sequenced, and sequence data were deposited in GenBank (MK249746). The query sequence was 99% identical to P. ambiguum (FR748048.1) in the NCBI database. The pathogenicity was confirmed in an open-air enclosure from March to July of 2018. In order to enhance root infection by this soil-borne fungus, 4-day-old seedlings of the hybrid MO1501 (Monsanto Spain SL) were used as plant material. They were transplanted in 5-liter pots filled with sand/silt/peat moss (2:1:2) (SSP) homogenously infested with P. ambiguum (Biota-2017 isolate) colonized wheat grains (Ortiz-Bustos et al. 2016). Seedlings of the controls were transplanted in noninfested SSP. In a different group of plants, another inoculation method was assayed by immersing a toothpick in a suspension of 10⁶ conidia/ml or in deionized water (control) for 6 h and then inserting it into the third internode of each plant in the tasseling stage (Aguiar et al. 2016). For each inoculation method, the experimental unit consisted of one plant (pot), and six replications were established for each treatment according to a complete randomized design. Greater percent leaf senescence (LS) per plant was observed 9 weeks after transplanting in infested soil (11.7% compared with 4.2% in the controls) (P = 0.0276). At the end of the experiment, significant differences (sd) of LS were again observed: 55% in inoculated maize compared with 30.1% in the controls (P < 0.0001), as well as sd of height (P = 0.0050) (178 and 242 cm in inoculated and control plants, respectively). With toothpick insertion, sd of LS only occurred at the final time (30.8 and 21.7% in inoculated and control plants, respectively). Symptoms were reproduced with either inoculation method, but after transplant to infested soil, they appeared sooner and were greater at the end of the experiment. The fungus was reisolated from tissues of the first internode of three plants inoculated by each method, fulfilling Koch’s postulates. In Europe, P. ambiguum has been reported as causing stalk rot of maize in Bulgaria and France (Farr and Rossman 2019), but this is the first report on the pathogen’s shift to warmer areas (Spain and Portugal) and suggests that appropriate measures must be taken for controlling the disease.