Since its emergence in 2013, the H7N9 low pathogenic avian influenza virus (LPAIV) has been circulating in domestic poultry in China, causing five waves of human infections. Recently, a novel H7N9 highly pathogenic avian influenza virus (HPAIV) variant possessing multiple basic amino acids at the cleavage site of the hemagglutinin protein was firstly reported in two human infection cases in January 2017. More seriously, those novel HPAI H7N9 variants have transmitted and caused poultry farms outbreaks in eight provinces. Herein, we demonstrate the presence of three different amino acid motifs at the cleavage sites of these HPAIV variants which were isolated from chickens and humans and likely evolved from the pre-existing LPAIVs. Animal experiments showed that these novel HPAI H7N9 variants are both highly pathogenic in chickens and lethal to mice. Notably, human origin viruses were more pathogenic in mice than avian viruses, and the mammalian adaptation associated E627K, A588V, and D701N mutations in the PB2 gene were identified in the infected mice using next-generation sequencing (NGS) and Sanger sequencing. In the key amino acid substitutions of PB2 and HA, no polymorphism was detected in the infected chickens lungs by NGS. In sum, these results highlight the highly pathogenicity and transmission in chickens, and the quickly adaptation in mammals of this new H7N9 variant, so the risk should be evaluated and payed more attention.IMPORTANCE Due to the recent increase zoonotic infections in poultry and persistent human infections in China, influenza A (H7N9) virus has remained a public health threat. Most of the A(H7N9) viruses previously reported have been of low pathogenicity. Now, these novel H7N9 HPAIV have caused human infection in three provinces and poultry farms outbreaks in eight provinces. We analyze these molecular features and compared the relative characteristics of one H7N9 LPAIV and two H7N9 HPAIVs isolated from chickens and two human-origin H7N9 HPAIVs in chicken and mice models. Our finding that all HPAIVs are both highly pathogenic and valid transmissibility in chickens. Strikingly, the human-origin viruses were more highly pathogenic than avian-origin viruses in mice, dynamic mutation were confirmed by NGS and Sanger sequencing. Our findings offer important insight into the origin, adaptation, pathogenicity, transmissibility to both poultry and mammals.