Spinal muscular atrophy (SMA) is an inherited neuromuscular disease characterized by progressive skeletal muscular weakness and atrophy. The newborn screening for spinal muscular atrophy should define all molecular forms of SMA. The aim of this study is to compare a PCR-based test for detection of homozygous SMN1 loss with multiple ligation probe amplification (MPLA) in patients with spinal muscular atrophy and other numerical changes of the SMN1 gene. PCR-based test was used to detect exon 7 of SMN1 gene homozygous loss. The study included 341 samples of patients with clinical suspicion of SMA from Biobank of Centre of Molecular Medicine of Pavlov State Medical University (Saint-Petersburg, Russia). Group 1 included 206 whole blood samples and Group 2 included 135 dried blood spot (DBS) samples. Copy number of the SMN1 and SMN2 genes had been evaluated with MLPA as a reference method. The results showed that kit was able to detect homozygous SMN1 loss in all samples from group 1 and 2 (Group 1: n = 67; 33%; Group 2: n = 19; 14%). At the same time in all samples with 1–3 copies of the SMN1 gene, the results of the kit were negative for homozygous loss of SMN1 gene (Group 1: n = 139; 67%; Group 2: n = 116; 86%). Kit showed high effectiveness in the detection of homozygous loss SMN1 gene. The kit detects all possible molecular forms of homozygous SMN1 gene loss in both DNA samples extracted from the whole blood and DBS.