Human infections with avian influenza viruses are a serious public health concern. The neuraminidase (NA) inhibitors (NAIs) are the frontline anti-influenza drugs and are the major option for treatment of newly emerging influenza. Therefore, it is essential to identify the molecular markers of NAI-resistance among specific NA subtypes of avian influenza viruses to help guide clinical management. NAI-resistant substitutions in NA subtypes other than N1 and N2 have been poorly studied. Here we identified NA amino acid substitutions associated with NAI resistance among influenza viruses of N3, N7 and N9 subtypes which have been associated with zoonotic transmission. We applied random mutagenesis and generated recombinant influenza viruses carrying single or double NA substitution(s) with 7 internal genes from A/Puerto Rico/8/1934 (H1N1) virus. In fluorescence-based NA inhibition assay we identified 3 categories of NA substitutions associated with reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir); i) novel subtype-specific substitutions in or near the enzyme catalytic site (R152W, A246T, and D293N, N2 numbering); ii) subtype-independent substitutions (E119G/V and/or D and R292K); and iii) substitutions previously reported in other subtypes (Q136K, I222M, and E276D). Our data show that although some markers of resistance are present across NA subtypes, other subtype-specific markers can only be determined empirically.