Piscirickettsiosis (SRS), caused by Piscirickettsia salmonis, is the main infectious disease of farmed Atlantic salmon in Chile. The current official surveillance and control plan for SRS has been based only on the detection of P. salmonis, but not on its genogroups (LF-89-like and EM-90-like). Surveillance at the genogroup level is essential not only for defining and evaluating the vaccination strategy against SRS, but is also of utmost importance for early diagnosis, clinical prognosis in the field, treatment and control of the disease. The objectives of this study were to characterize the spatiotemporal distribution of P. salmonis genogroups using genogroup-specific qPCR to discriminate LF-89-like and EM-90-like within and between seawater farms, individual fish, and tissues/organs during early infection in Atlantic salmon under field conditions. The spatiotemporal distribution of LF-89-like and EM-90-like was highly variable within and between seawater farms. P. salmonis infection is multigenogroup at the farm, fish and tissue levels. Our study demonstrated for the first time a complex co-infection of P. salmonis LF-89-like and EM-90-like in Atlantic salmon. Liver nodules could be associated with EM-90-like infection, but not with LF-89-like or co-infection of both genogroups. The positivity rate of P. salmonis LF-89-like increased significantly between 2017 and 2021 and was the main circulating genogroup in Chilean salmon aquaculture during this period (Figure 1). Finally, a strategy to identify P. salmonis genogroups based on novel genogroup-specific qPCR for LF-89-like and EM-90-like genogroups was reported. Consequently, our results help to better understand the biological interaction of P. salmonis and the host and generate knowledge to improve the surveillance and control strategy for SRS in Chile.