Tomato (Lycopersicon esculentum Mill) is a highly consumed and produced vegetable crop with an annual global production of over 185 million tonnes. High consumption and production are due to its nutritional and health benefits. However, tomato production is characterized by inconsistent quality and yields, partly emanating from biotic constraints attributed to fungal foliar diseases. In Kirinyaga County, where tomato production is a significant source of livelihood, knowledge gaps exist regarding the identity of Alternaria species causing tomato blight in different Agro-ecological zones (AEZs) and the susceptibility of locally grown tomato varieties to the Alternaria pathogens. To address these gaps, we conducted a study to determine the characteristics of an Alternaria pathogen isolated from tomato leaves from different AEZs and assess the susceptibility level of some tomato varieties grown in Kirinyaga County. Pathogen characterization was done using phenotypic and molecular approaches, and the susceptibility assay was evaluated by artificial inoculation. The molecular characterization involved BLASTIN analysis of Sanger sequenced DNA nucleotide amplicons, and the use of NCBI databases. The susceptibility assay was carried out in the greenhouse using a completely randomized design. Data on the fungal conidial lengths (µm) and the susceptibility (%) of tomato varieties to fungal isolates were subjected to Analysis of Variance (ANOVA) with significance means separated using LSD at α = 0.05. The morphologies of the pathogens differed significantly (p <0.05). Molecular characterization implicated Alternaria solani, Alternaria cerealis, Alternaria arborescens, and Alternaria alternate in causing tomato blight. Tomato varieties differed in their susceptibility to Alternaria pathogens (p < 0.05). Kilele F1 (73.84%), and Terminator F1 (56.48%) were the most and least susceptible tomato varieties, respectively. Thus, Terminator F1 can serve as a source for the genetic improvement of tomato varieties in the Kirinyaga patho-system. Our study provides valuable information on the identification of Alternaria species causing tomato blight in different AEZs in Kirinyaga County and the susceptibility of locally grown tomato varieties to infection by Alternaria pathogen isolates. The findings can aid in the development of effective disease management strategies and the genetic improvement of tomato varieties in the area.