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• The peak plasma ionospheric frequency (f o F2) from ionosonde stations located at Islamabad and Sonmiani is analyzed. • The 30 days running median technique to detect the abnormality in f o F2 over the epicenter of impending earthquake. • The major reason of these seismo-ionospheric anomalies is the distance between the ionosonde station and epicenter. • The evidence supports that these seismo-ionospheric precursors are probably due to the lithospheric-ionospheric coupling. The rapid advancement in ground and space based ionospheric measurements provide an opportunity to work on different earthquake precursors for lithosphere-ionosphere coupling hypothesis. In this paper, the peak plasma ionospheric frequency (f o F2) for 90 days before/after the main shock of September 24, 2019 (M5.6) earthquake in Pakistan are studied for earthquake precursors from ionosonde stations located at Islamabad and Sonmiani. We implement the 30 days running median technique to detect the abnormality in f o F2 over the epicenter of impending earthquake. A comprehensive analysis of these anomalies on two stations is performed in order to extract the maximum of these abnormalities in their respective regions. The deviation in hourly data in Sonmiani station shows significant variation within 10–20 days before the main shock, as most of the values within 5–10 days' window are within the confidence limits. On the other hand, positive and negative deviations in the analysis of Islamabad station may be revealed as a possible signature of seismo-ionospheric anomalies. The major reason of these seismo-ionospheric anomalies is the distance between the ionosonde station and epicenter, where Islamabad station is close to the epicenter as compared to Sonmiani. It is noteworthy that both negative and positive deviations are observed before the M5.6 earthquake; however, the intensity of positive anomalies is more than negative anomalies. Moreover, severe positive deviation occurs within 10–20 days before the earthquake at the Sonmiani station. Also, there is no geomagnetic storm within 10 days before the earthquake which opposes the existence of seismo-ionospheric anomalies. The evidence supports that these seismo-ionospheric precursors are probably due to the lithospheric-ionospheric coupling. [ABSTRACT FROM AUTHOR]