Swine influenza virus (SIV) is widespread in pigs, complicating vaccination efforts due to phenotypic diversity among isolates. This study examined 170 SIV isolates from 103 farms in Brazil, with 134 (78.8%) categorized into various subtypes using a multiplex polymerase chain reaction (mPCR) assay: the most prevalent subtypes were H1pdmN1pdm (44/32.8%); H1pdmN2 (33/24.6%); H1huN2 (41/30.6%); H1huN1pdm (4/3%); and H3N2 (12/9%), while 36 (21.2%) were non-typeable (NT) by mPCR. One isolate from each subtype was used in mono- or polyvalent vaccines for immunizing rats and pigs and to assess the cross-reactivity of the serum using the hemagglutination inhibition assay (HI) against 99 randomly assigned isolates. Among these, there were 29 H1hu isolates, of which 23 (79.3%) were identified by rat monospecific serum and 22 (75.8%) by rat and swine polyspecific serum. Among the 30 H1pdm subtypes (36.4% of the tested isolates), 14 (46.6%), 16 (53.3%), and 13 (43.3%) were recognized by the homologous serum; and, among the 5 H3N2 isolates (5.0% of the isolates), the recognition rates were 3 (60%), 4 (80%), and 3 (60%), respectively. Our results indicated that viral subtypes are widely distributed across Brazil, and a significant proportion of the isolates were identified by serum generated by the polyvalent vaccines. The vaccines elicit antibodies that could recognize a large proportion, but not all isolates. This work supports the use of rats to assess SIV immunogenicity, given consistency in cross-reactivity results obtained with serum from both species.