The Trans-Mexican Volcanic Belt (TMVB) is a calc-alkaline volcanic arc cut by different active crustal fault systems that have originated several destructive historical earthquakes. Located in the central part of Mexico this region offers exceptional climatic, and fertility of soil conditions, which is the reason why more than 50% of the Mexican population now live here, increasing the seismic risk. Determining the seismic potential of these fault systems is important in the western section of the TMVB, in the vicinity of the city of Guadalajara, where more than 5 million inhabitants are concentrated in a densely populated urban area.We focus here on the epicentral area of the MW 7.2 sixteenth century Ameca earthquake, one of the first earthquakes described to take place in the American continent and which also may be the largest crustal earthquake to have occurred in the TMVB in the historical record. According to some historical sources, this earthquake would be associated with the Ameca-Ahuisculco Fault but no neotectonic study has been carried out so far to characterize this fault. Here, we describe the geomorphology of the fault escarpment and the characteristics of different fault segments. This first step allowed to select a suitable site for a paleoseismological study to track the historic event. The results of the interpretation of two trenches are consistent, showing evidence of net activity of the fault in the tectono-sedimentary record with two and possibly three seismic events. The older one of these is not well recorded and interpreted as a possible event that could have occurred after 27,91 ± 0,4 cal ka BP and before 5,67 ± 0,064 cal ka BP. The second one and best recorded event occurred around 5,67 ± 0,064 cal ka BP whilst the last one occurred after 0,985 ± 0,065 cal ka BP and is likely to be the geological record of the Ameca sixteenth century earthquake. Considering the potential rupture lengths and the coseismic displacement measured in the trenches, this fault system seems capable of generating earthquakes of magnitude 6.9 to 7.3 and represents a major source of earthquake hazard to the city of Guadalajara.